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Enzymes that form filamentous assemblies with modulated enzymatic activities have gained increasing attention in recent years. SgrAI is a sequence specific type II restriction endonuclease that forms polymeric filaments with accelerated DNA cleavage activity and expanded DNA sequence specificity. Prior studies have suggested a mechanistic model linking the structural changes accompanying SgrAI filamentation to its accelerated DNA cleavage activity. In this model, the conformational changes that are specific to filamentous SgrAI maximize contacts between different copies of the enzyme within the filament and create a second divalent cation binding site in each subunit, which in turn facilitates the DNA cleavage reaction. However, our understanding of the atomic mechanism of catalysis is incomplete. Herein, we present two new structures of filamentous SgrAI solved using cryo-EM. The first structure, resolved to 3.3 Å, is of filamentous SgrAI containing an active site mutation that is designed to stall the DNA cleavage reaction, which reveals the enzymatic configuration prior to DNA cleavage. The second structure, resolved to 3.1 Å, is of WT filamentous SgrAI containing cleaved substrate DNA, which reveals the enzymatic configuration at the end of the enzymatic cleavage reaction. Both structures contain the phosphate moiety at the cleavage site and the biologically relevant divalent cation cofactor Mg 2+ and define how the Mg 2+ cation reconfigures during enzymatic catalysis. The data support a model for the activation mechanism that involves binding of a second Mg 2+ in the SgrAI active site as a direct result of filamentation induced conformational changes., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The therapeutic application of CRISPR-Cas9 is limited due to its off-target activity. To have a better understanding of this off-target effect, we focused on its mismatch-prone PAM distal end. The off-target activity of SpCas9 depends directly on the nature of mismatches, which in turn results in deviation of the active site of SpCas9 due to structural instability in the RNA-DNA duplex strand. In order to test the hypothesis, we designed an array of mismatched target sites at the PAM distal end and performed in vitro and cell line-based experiments, which showed a strong correlation for Cas9 activity. We found that target sites having multiple mismatches in the 18th to 15th position upstream of the PAM showed no to little activity. For further mechanistic validation, Molecular Dynamics simulations were performed, which revealed that certain mismatches showed elevated root mean square deviation values that can be attributed to conformational instability within the RNA-DNA duplex. Therefore, for successful prediction of the off-target effect of SpCas9, along with complementation-derived energy, the RNA-DNA duplex stability should be taken into account., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

AT-rich interacting domain (ARID)-containing proteins, Arids, are a heterogeneous DNA-binding protein family involved in transcription regulation and chromatin processing. For the member Arid5a, no exact DNA-binding preference has been experimentally defined so far. Additionally, the protein binds to mRNA motifs for transcript stabilization, supposedly through the DNA-binding ARID domain. To date, however, no unbiased RNA motif definition and clear dissection of nucleic acid-binding through the ARID domain have been undertaken. Using NMR-centered biochemistry, we here define the Arid5a DNA preference. Further, high-throughput in vitro binding reveals a consensus RNA-binding motif engaged by the core ARID domain. Finally, transcriptome-wide binding (iCLIP2) reveals that Arid5a has a weak preference for (A)U-rich regions in pre-mRNA transcripts of factors related to RNA processing. We find that the intrinsically disordered regions flanking the ARID domain modulate the specificity and affinity of DNA binding, while they appear crucial for RNA interactions. Ultimately, our data suggest that Arid5a uses its extended ARID domain for bifunctional gene regulation and that the involvement of IDR extensions is a more general feature of Arids in interacting with different nucleic acids at the chromatin-mRNA interface., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Over the past decade, the connection between APOBEC3 cytosine deaminases and cancer mutagenesis has become increasingly apparent. This growing awareness has created a need for biochemical tools that can be used to identify and characterize potential inhibitors of this enzyme family. In response to this challenge, we have developed a Real-time APOBEC3-mediated DNA Deamination assay. This assay offers a single-step set-up and real-time fluorescent read-out, and it is capable of providing insights into enzyme kinetics. The assay also offers a high-sensitivity and easily scalable method for identifying APOBEC3 inhibitors. This assay serves as a crucial addition to the existing APOBEC3 biochemical and cellular toolkit and possesses the versatility to be readily adapted into a high-throughput format for inhibitor discovery., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Nucleoside analogue drugs are pervasively used as antiviral and chemotherapy agents. Cytarabine and gemcitabine are anti-cancer nucleoside analogue drugs that contain C2' modifications on the sugar ring. Despite carrying all the required functional groups for DNA synthesis, these two compounds inhibit DNA extension once incorporated into DNA. It remains unclear how the C2' modifications on cytarabine and gemcitabine affect the polymerase active site during substrate binding and DNA extension. Using steady-state kinetics, static and time-resolved X-ray crystallography with DNA polymerase η (Pol η) as a model system, we showed that the sugar ring C2' chemical groups on cytarabine and gemcitabine snugly fit within the Pol η active site without occluding the steric gate. During DNA extension, Pol η can extend past gemcitabine but with much lower efficiency past cytarabine. The Pol η crystal structures show that the -OH modification in the β direction on cytarabine locks the sugar ring in an unfavorable C2'-endo geometry for product formation. On the other hand, the addition of fluorine atoms on gemcitabine alters the proper conformational transition of the sugar ring for DNA synthesis. Our study illustrates mechanistic insights into chemotherapeutic drug inhibition and resistance and guides future optimization of nucleoside analogue drugs., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Human DNA ligase 1 (LIG1) is the main replicative ligase that seals Okazaki fragments during nuclear replication and finalizes DNA repair pathways by joining DNA ends of the broken strand breaks in the three steps of the ligation reaction. LIG1 can tolerate the RNA strand upstream of the nick, yet an atomic insight into the sugar discrimination mechanism by LIG1 against a ribonucleotide at the 3'-terminus of nick DNA is unknown. Here, we determined X-ray structures of LIG1/3'-RNA-DNA hybrids and captured the ligase during pre- and post-step 3 the ligation reaction. Furthermore, the overlays of 3'-rA:T and 3'-rG:C step 3 structures with step 2 structures of canonical 3'-dA:T and 3'-dG:C uncover a network of LIG1/DNA interactions through Asp570 and Arg871 side chains with 2'-OH of the ribose at nick showing a final phosphodiester bond formation and the other ligase active site residues surrounding the AMP site. Finally, we demonstrated that LIG1 can ligate the nick DNA substrates with pre-inserted 3'-ribonucleotides as efficiently as Watson-Crick base-paired ends in vitro. Together, our findings uncover a novel atomic insight into a lack of sugar discrimination by LIG1 and the impact of improper sugar on the nick sealing of ribonucleotides at the last step of DNA replication and repair., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Histone proteins can become trapped on DNA in the presence of 5-formylcytosine (5fC) to form toxic DNA-protein conjugates. Their repair may involve proteolytic digestion resulting in DNA-peptide cross-links (DpCs). Here, we have investigated replication of a model DpC comprised of an 11-mer peptide (NH 2 -GGGKGLGK∗GGA) containing an oxy-lysine residue (K∗) conjugated to 5fC in DNA. Both CXG and CXT (where X = 5fC-DpC) sequence contexts were examined. Replication of both constructs gave low viability (<10%) in Escherichia coli, whereas TLS efficiency was high (72%) in HEK 293T cells. In E. coli, the DpC was bypassed largely error-free, inducing only 2 to 3% mutations, which increased to 4 to 5% with SOS. For both sequences, semi-targeted mutations were dominant, and for CXG, the predominant mutations were G→T and G→C at the 3'-base to the 5fC-DpC. In HEK 293T cells, 7 to 9% mutations occurred, and the dominant mutations were the semi-targeted G → T for CXG and T → G for CXT. These mutations were reduced drastically in cells deficient in hPol η, hPol ι or hPol ζ, suggesting a role of these TLS polymerases in mutagenic TLS. Steady-state kinetics studies using hPol η confirmed that this polymerase induces G → T and T → G transversions at the base immediately 3' to the DpC. This study reveals a unique replication pattern of 5fC-conjugated DpCs, which are bypassed largely error-free in both E. coli and human cells and induce mostly semi-targeted mutations at the 3' position to the lesion., Competing Interests: Conflict of interest The authors declare no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The past 4 decades have witnessed tremendous efforts in deciphering the role of O-GlcNAcylation in a plethora of biological processes. Chemists and biologists have joined hand in hand in the sweet adventure to unravel this unique and universal yet uncharted post-translational modification, and the recent advent of cutting-edge chemical biology and mass spectrometry tools has greatly facilitated the process. Compared with O-GlcNAc, DNA damage response (DDR) is a relatively intensively studied area that could be traced to before the elucidation of the structure of DNA. Unexpectedly, yet somewhat expectedly, O-GlcNAc has been found to regulate various DDR pathways: homologous recombination, nonhomologous end joining, base excision repair, and translesion DNA synthesis. In this review, we first cover the recent structural studies of the O-GlcNAc transferase and O-GlcNAcase, the elegant duo that "writes" and "erases" O-GlcNAc modification. Then we delineate the intricate roles of O-GlcNAc transferase and O-GlcNAcase in DDR. We envision that this is only the beginning of our full appreciation of how O-GlcNAc regulates the blueprint of life-DNA., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

DNA modifications add another layer of complexity to the eukaryotic genome to regulate gene expression, playing critical roles as epigenetic marks. In eukaryotes, the study of DNA epigenetic modifications has been confined to 5mC and its derivatives for decades. However, rapid developing approaches have witnessed the expansion of DNA modification reservoirs during the past several years, including the identification of 6mA, 5gmC, 4mC, and 4acC in diverse organisms. However, whether these DNA modifications function as epigenetic marks requires careful consideration. In this review, we try to present a panorama of all the DNA epigenetic modifications in eukaryotes, emphasizing recent breakthroughs in the identification of novel DNA modifications. The characterization of their roles in transcriptional regulation as potential epigenetic marks is summarized. More importantly, the pathways for generating or eliminating these DNA modifications, as well as the proteins involved are comprehensively dissected. Furthermore, we briefly discuss the potential challenges and perspectives, which should be taken into account while investigating novel DNA modifications., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Mutations in, or deficiency of, fragile X messenger ribonucleoprotein (FMRP) is responsible for the Fragile X syndrome (FXS), the most common cause for inherited intellectual disability. FMRP is a nucleocytoplasmic protein, primarily characterized as a translation repressor with poorly understood nuclear function(s). We recently reported that FXS patient cells lacking FMRP sustain higher level of DNA double-strand breaks (DSBs) than normal cells, specifically at sequences prone to forming R-loops, a phenotype further exacerbated by DNA replication stress. Moreover, expression of FMRP, and not an FMRPI304N mutant known to cause FXS, reduced R-loop-associated DSBs. We subsequently reported that recombinant FMRP directly binds R-loops, primarily through the carboxyl terminal intrinsically disordered region. Here, we show that FMRP directly interacts with an RNA helicase, DHX9. This interaction, which is mediated by the amino terminal structured domain of FMRP, is reduced with FMRPI304N. We also show that FMRP inhibits DHX9 helicase activity on RNA:DNA hybrids and the inhibition is also dependent on the amino terminus. Furthermore, the FMRPI304N mutation causes both FMRP and DHX9 to persist on the chromatin in replication stress. These results suggest an antagonistic relationship between FMRP and DHX9 at the chromatin, where their proper interaction leads to dissociation of both proteins from the fully resolved R-loop. We propose that the absence or the loss of function of FMRP leads to persistent presence of DHX9 or both proteins, respectively, on the unresolved R-loop, ultimately leading to DSBs. Our study sheds new light on our understanding of the genome functions of FMRP., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Nucleoid-associated proteins (NAPs) regulate multiple cellular processes such as gene expression, virulence, and dormancy throughout bacterial species. NAPs help in the survival and adaptation of Mycobacterium tuberculosis (Mtb) within the host. Fourteen NAPs have been identified in Escherichia coli; however, only seven NAPs are documented in Mtb. Given its complex lifestyle, it is reasonable to assume that Mtb would encode for more NAPs. Using bioinformatics tools and biochemical experiments, we have identified the heparin-binding hemagglutinin (HbhA) protein of Mtb as a novel sequence-independent DNA-binding protein which has previously been characterized as an adhesion molecule required for extrapulmonary dissemination. Deleting the carboxy-terminal domain of HbhA resulted in a complete loss of its DNA-binding activity. Atomic force microscopy showed HbhA-mediated architectural modulations in the DNA, which may play a regulatory role in transcription and genome organization. Our results showed that HbhA colocalizes with the nucleoid region of Mtb. Transcriptomics analyses of a hbhA KO strain revealed that it regulates the expression of ∼36% of total and ∼29% of essential genes. Deletion of hbhA resulted in the upregulation of ∼73% of all differentially expressed genes, belonging to multiple pathways suggesting it to be a global repressor. The results show that HbhA is a nonessential NAP regulating gene expression globally and acting as a plausible transcriptional repressor., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

3D chromatin organization plays a critical role in regulating gene expression, DNA replication, recombination, and repair. While initially discovered for its role in sister chromatid cohesion, emerging evidence suggests that the cohesin complex (SMC1, SMC3, RAD21, and SA1/SA2), facilitated by NIPBL, mediates topologically associating domains and chromatin loops through DNA loop extrusion. However, information on how conformational changes of cohesin-NIPBL drive its loading onto DNA, initiation, and growth of DNA loops is still lacking. In this study, high-speed atomic force microscopy imaging reveals that cohesin-NIPBL captures DNA through arm extension, assisted by feet (shorter protrusions), and followed by transfer of DNA to its lower compartment (SMC heads, RAD21, SA1, and NIPBL). While binding at the lower compartment, arm extension leads to the capture of a second DNA segment and the initiation of a DNA loop that is independent of ATP hydrolysis. The feet are likely contributed by the C-terminal domains of SA1 and NIPBL and can transiently bind to DNA to facilitate the loading of the cohesin complex onto DNA. Furthermore, high-speed atomic force microscopy imaging reveals distinct forward and reverse DNA loop extrusion steps by cohesin-NIPBL. These results advance our understanding of cohesin by establishing direct experimental evidence for a multistep DNA-binding mechanism mediated by dynamic protein conformational changes., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.Figure Legends, (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Alanyl-tRNA synthetase retains a conserved prototype structure throughout its biology. Nevertheless, its C-terminal domain (C-Ala) is highly diverged and has been shown to play a role in either tRNA or DNA binding. Interestingly, we discovered that Caenorhabditis elegans cytoplasmic C-Ala (Ce-C-Ala c ) robustly binds both ligands. How Ce-C-Ala c targets its cognate tRNA and whether a similar feature is conserved in its mitochondrial counterpart remain elusive. We show that the N- and C-terminal subdomains of Ce-C-Ala c are responsible for DNA and tRNA binding, respectively. Ce-C-Ala c specifically recognized the conserved invariant base G 18 in the D-loop of tRNA Ala through a highly conserved lysine residue, K934. Despite bearing little resemblance to other C-Ala domains, C. elegans mitochondrial C-Ala robustly bound both tRNA Ala and DNA and maintained targeting specificity for the D-loop of its cognate tRNA. This study uncovers the underlying mechanism of how C. elegans C-Ala specifically targets the D-loop of tRNA Ala ., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The role of alternate DNA conformations such as Z-DNA in the regulation of transcription is currently underappreciated. These structures are encoded by sequences called flipons, many of which are enriched in promoter and enhancer regions. Through a change in their conformation, flipons provide a tunable mechanism to mechanically reset promoters for the next round of transcription. They act as actuators that capture and release energy to ensure that the turnover of the proteins at promoters is optimized to cell state. Likewise, the single-stranded DNA formed as flipons cycle facilitates the docking of RNAs that are able to microcode promoter conformations and canalize the pervasive transcription commonly observed in metazoan genomes. The strand-specific nature of the interaction between RNA and DNA likely accounts for the known asymmetry of epigenetic marks present on the histone tetramers that pair to form nucleosomes. The role of these supercoil-dependent processes in promoter choice and transcriptional interference is reviewed. The evolutionary implications are examined: the resilience and canalization of flipon-dependent gene regulation is contrasted with the rapid adaptation enabled by the spread of flipon repeats throughout the genome. Overall, the current findings underscore the important role of flipons in modulating the readout of genetic information and how little we know about their biology., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

CRISPR-Cas adaptive immune systems uptake short "spacer" sequences from foreign DNA and incorporate them into the host genome to serve as templates for CRISPR RNAs that guide interference against future infections. Adaptation in CRISPR systems is mediated by Cas1-Cas2 complexes that catalyze integration of prespacer substrates into the CRISPR array. Many DNA targeting systems also require Cas4 endonucleases for functional spacer acquisition. Cas4 selects prespacers containing a protospacer adjacent motif (PAM) and removes the PAM prior to integration, both of which are required to ensure host immunization. Cas1 has also been shown to function as a nuclease in some systems, but a role for this nuclease activity in adaptation has not been demonstrated. We identified a type I-G Cas4/1 fusion with a nucleolytically active Cas1 domain that can directly participate in prespacer processing. The Cas1 domain is both an integrase and a sequence-independent nuclease that cleaves the non-PAM end of a prespacer, generating optimal overhang lengths that enable integration at the leader side. The Cas4 domain sequence specifically cleaves the PAM end of the prespacer, ensuring integration of the PAM end at the spacer side. The two domains have varying metal ion requirements. While Cas4 activity is Mn 2+ dependent, Cas1 preferentially uses Mg 2+ over Mn 2+ . The dual nuclease activity of Cas4/1 eliminates the need for additional factors in prespacer processing making the adaptation module self-reliant for prespacer maturation and directional integration., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The DNA adduct 6-oxo-M 1 dG, (3-(2'-deoxy-β-D-erythro-pentofuranosyl)-6-oxo-pyrimido(1,2alpha)purin-10(3H)-one) is formed in the genome via oxidation of the peroxidation-derived adduct M 1 dG. However, the effect of 6-oxo-M 1 dG adducts on subsequent DNA replication is unclear. Here we investigated the ability of the human Y-family polymerase hPol η to bypass 6-oxo-M 1 dG. Using steady-state kinetics and analysis of DNA extension products by liquid chromatography-tandem mass spectrometry, we found hPol η preferentially inserts a dAMP or dGMP nucleotide into primer-templates across from the 6-oxo-M 1 dG adduct, with dGMP being slightly preferred. We also show primer-templates with a 3'-terminal dGMP or dAMP across from 6-oxo-M 1 dG were extended to a greater degree than primers with a dCMP or dTMP across from the adduct. In addition, we explored the structural basis for bypass of 6-oxo-M 1 dG by hPol η using X-ray crystallography of both an insertion-stage and an extension-stage complex. In the insertion-stage complex, we observed that the incoming dCTP opposite 6-oxo-M 1 dG, although present during crystallization, was not present in the active site. We found the adduct does not interact with residues in the hPol η active site but rather forms stacking interactions with the base pair immediately 3' to the adduct. In the extension-stage complex, we observed the 3' hydroxyl group of the primer strand dGMP across from 6-oxo-M 1 dG is not positioned correctly to form a phosphodiester bond with the incoming dCTP. Taken together, these results indicate 6-oxo-M 1 dG forms a strong block to DNA replication by hPol η and provide a structural basis for its blocking ability., Competing Interests: Conflict of interest The authors declare no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Force and torque spectroscopy have provided unprecedented insights into the mechanical properties, conformational transitions, and dynamics of DNA and DNA-protein complexes, notably nucleosomes. Reliable single-molecule manipulation measurements require, however, specific and stable attachment chemistries to tether the molecules of interest. Here, we present a functionalization strategy for DNA that enables high-yield production of constructs for torsionally constrained and very stable attachment. The method is based on two subsequent PCRs: first ∼380 bp long DNA strands are generated that contain multiple labels, which are used as "megaprimers" in a second PCR to generate ∼kbp long double-stranded DNA constructs with multiple labels at the respective ends. To achieve high-force stability, we use dibenzocyclooctyne-based click chemistry for covalent attachment to the surface and biotin-streptavidin coupling to the bead. The resulting tethers are torsionally constrained and extremely stable under load, with an average lifetime of 70 ± 3 h at 45 pN. The high yield of the approach enables nucleosome reconstitution by salt dialysis on the functionalized DNA, and we demonstrate proof-of-concept measurements on nucleosome assembly statistics and inner turn unwrapping under force. We anticipate that our approach will facilitate a range of studies of DNA interactions and nucleoprotein complexes under forces and torques., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Thymine DNA glycosylase (TDG) is an essential enzyme involved in numerous biological pathways, including DNA repair, DNA demethylation, and transcriptional activation. Despite these important functions, the mechanisms surrounding the actions and regulation of TDG are poorly understood. In this study, we demonstrate that TDG induces phase separation of DNA and nucleosome arrays under physiologically relevant conditions in vitro and show that the resulting chromatin droplets exhibited behaviors typical of phase-separated liquids, supporting a liquid-liquid phase separation model. We also provide evidence that TDG has the capacity to form phase-separated condensates in the cell nucleus. The ability of TDG to induce chromatin phase separation is dependent on its intrinsically disordered N- and C-terminal domains, which in isolation, promote the formation of chromatin-containing droplets having distinct physical properties, consistent with their unique mechanistic roles in the phase separation process. Interestingly, DNA methylation alters the phase behavior of the disordered domains of TDG and compromises formation of chromatin condensates by full-length TDG, indicating that DNA methylation regulates the assembly and coalescence of TDG-mediated condensates. Overall, our results shed new light on the formation and physical nature of TDG-mediated chromatin condensates, which have broad implications for the mechanism and regulation of TDG and its associated genomic processes., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The BEN domain-containing transcription factors regulate transcription by recruiting chromatin-modifying factors to specific chromatin regions via their DNA-binding BEN domains. The BEN domain of BANP has been shown to bind to a CGCG DNA sequence or an AAA-containing matrix attachment regions DNA sequence. Consistent with these in vivo observations, we identified an optimal DNA-binding sequence of AAATCTCG by protein binding microarray, which was also confirmed by our isothermal titration calorimetry and mutagenesis results. We then determined crystal structures of the BANP BEN domain in apo form and in complex with a CGCG-containing DNA, respectively, which revealed that the BANP BEN domain mainly used the electrostatic interactions to bind DNA with some base-specific interactions with the TC motifs. Our isothermal titration calorimetry results also showed that BANP bound to unmethylated and methylated DNAs with comparable binding affinities. Our complex structure of BANP-mCGCG revealed that the BANP BEN domain bound to the unmethylated and methylated DNAs in a similar mode and cytosine methylation did not get involved in binding, which is also consistent with our observations from the complex structures of the BEND6 BEN domain with the CGCG or CGmCG DNAs. Taken together, our results further elucidate the elements important for DNA recognition and transcriptional regulation by the BANP BEN domain-containing transcription factor., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

For cells, it is important to repair DNA damage, such as double-strand and single-strand DNA breaks, because unrepaired DNA can compromise genetic integrity, potentially leading to cell death or cancer. Cells have multiple DNA damage repair pathways that have been the subject of detailed genetic, biochemical, and structural studies. Recently, the scientific community has started to gain evidence that the repair of DNA double-strand breaks may occur within biomolecular condensates and that condensates may also contribute to DNA damage through concentrating genotoxic agents used to treat various cancers. Here, we summarize key features of biomolecular condensates and note where they have been implicated in the repair of DNA double-strand breaks. We also describe evidence suggesting that condensates may be involved in the repair of other types of DNA damage, including single-strand DNA breaks, nucleotide modifications (e.g., mismatch and oxidized bases), and bulky lesions, among others. Finally, we discuss old and new mysteries that could now be addressed considering the properties of condensates, including chemoresistance mechanisms., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Photolyases (PLs) reverse UV-induced DNA damage using blue light as an energy source. Of these PLs, (6-4) PLs repair (6-4)-lesioned photoproducts. We recently identified a gene from Vibrio cholerae (Vc) encoding a (6-4) PL, but structural characterization is needed to elucidate specific interactions with the chromophore cofactors. Here, we determined the crystal structure of Vc (6-4) PL at 2.5 Å resolution. Our high-resolution structure revealed that the two well-known cofactors, flavin adenine dinucleotide and the photoantenna 6,7-dimethyl 8-ribityl-lumazin (DMRL), stably interact with an α-helical and an α/β domain, respectively. Additionally, the structure has a third cofactor with distinct electron clouds corresponding to a [4Fe-4S] cluster. Moreover, we identified that Asp106 makes a hydrogen bond with water and DMRL, which indicates further stabilization of the photoantenna DMRL within Vc (6-4) PL. Further analysis of the Vc (6-4) PL structure revealed a possible region responsible for DNA binding. The region located between residues 478 to 484 may bind the lesioned DNA, with Arg483 potentially forming a salt bridge with DNA to stabilize further the interaction of Vc (6-4) PL with its substrate. Our comparative analysis revealed that the DNA lesion could not bind to the Vc (6-4) PL in a similar fashion to the Drosophila melanogaster (Dm, (6-4)) PL without a significant conformational change of the protein. The 23rd helix of the bacterial (6-4) PLs seems to have remarkable plasticity, and conformational changes facilitate DNA binding. In conclusion, our structure provides further insight into DNA repair by a (6-4) PL containing three cofactors., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023. Published by Elsevier Inc.)

Unusual nucleic acid structures play vital roles as intermediates in many cellular processes and, in the case of peptide nucleic acid (PNA)-mediated triplexes, are leveraged as tools for therapeutic gene editing. However, due to their transient nature, an understanding of the factors that interact with and process dynamic nucleic acid structures remains limited. Here, we developed snapELISA (structure-specific nucleic acid-binding protein ELISA), a rapid high-throughput platform to interrogate and compare up to 2688 parallel nucleic acid structure-protein interactions in vitro. We applied this system to both triplex-forming oligonucleotide-induced DNA triplexes and DNA-bound PNA heterotriplexes to describe the identification of previously known and novel interactors for both structures. For PNA heterotriplex recognition analyses, snapELISA identified factors implicated in nucleotide excision repair (XPA, XPC), single-strand annealing repair (RAD52), and recombination intermediate structure binding (TOP3A, BLM, MUS81). We went on to validate selected factor localization to genome-targeted PNA structures within clinically relevant loci in human cells. Surprisingly, these results demonstrated XRCC5 localization to PNA triplex-forming sites in the genome, suggesting the presence of a double-strand break intermediate. These results describe a powerful comparative approach for identifying structure-specific nucleic acid interactions and expand our understanding of the mechanisms of triplex structure recognition and repair., Competing Interests: Conflict of interest P. M. G. and N. G. E. are inventors on patents related to PNAs and gene editing that are assigned to Yale University. P. M. G. is a consultant to and has equity in Cybrexa Therapeutics, pHLIP Inc, and Gennao Bio and has an equity interest in Patrys Limited. None of these entities were involved in or are related to the work reported here., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Specific DNA methylation at CpG and non-CpG sites is essential for chromatin regulation. The DNA methyltransferase DNMT3A interacts with target sites surrounded by variable DNA sequences with its TRD and RD loops, but the functional necessity of these interactions is unclear. We investigated CpG and non-CpG methylation in a randomized sequence context using WT DNMT3A and several DNMT3A variants containing mutations at DNA-interacting residues. Our data revealed that the flanking sequence of target sites between the -2 and up to the +8 position modulates methylation rates >100-fold. Non-CpG methylation flanking preferences were even stronger and favor C(+1). R836 and N838 in concert mediate recognition of the CpG guanine. R836 changes its conformation in a flanking sequence-dependent manner and either contacts the CpG guanine or the +1/+2 flank, thereby coupling the interaction with both sequence elements. R836 suppresses activity at CNT sites but supports methylation of CAC substrates, the preferred target for non-CpG methylation of DNMT3A in cells. N838 helps to balance this effect and prevent the preference for C(+1) from becoming too strong. Surprisingly, we found L883 reduces DNMT3A activity despite being highly conserved in evolution. However, mutations at L883 disrupt the DNMT3A-specific DNA interactions of the RD loop, leading to altered flanking sequence preferences. Similar effects occur after the R882H mutation in cancer cells. Our data reveal that DNMT3A forms flexible and interdependent interaction networks with the CpG guanine and flanking residues that ensure recognition of the CpG and efficient methylation of the cytosine in contexts of variable flanking sequences., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Many transcription factors contain intrinsically disordered transcription activation domains (TADs), which mediate interactions with coactivators to activate transcription. Historically, DNA-binding domains and TADs have been considered as modular units, but recent studies have shown that TADs can influence DNA binding. Whether these results can be generalized to more TADs is not clear. Here, we biophysically characterized the NFκB p50/RelA heterodimer including the RelA TAD and investigated the TAD's influence on NFκB-DNA interactions. In solution, we show the RelA TAD is disordered but compact, with helical tendency in two regions that interact with coactivators. We determined that the presence of the TAD increased the stoichiometry of NFκB-DNA complexes containing promoter DNA sequences with tandem κB recognition motifs by promoting the binding of NFκB dimers in excess of the number of κB sites. In addition, we measured the binding affinity of p50/RelA for DNA containing tandem κB sites and single κB sites. While the presence of the TAD enhanced the binding affinity of p50/RelA for all κB sequences tested, it also increased the affinity for nonspecific DNA sequences by over 10-fold, leading to an overall decrease in specificity for κB DNA sequences. In contrast, previous studies have generally reported that TADs decrease DNA-binding affinity and increase sequence specificity. Our results reveal a novel function of the RelA TAD in promoting binding to nonconsensus DNA, which sheds light on previous observations of extensive nonconsensus DNA binding by NFκB in vivo in response to strong inflammatory signals., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Apurinic/apyrimidinic (AP or abasic) sites are among the most abundant DNA lesions. Numerous proteins within different organisms ranging from bacteria to human have been demonstrated to react with AP sites to form covalent Schiff base DNA-protein cross-links (DPCs). These DPCs are unstable due to their spontaneous hydrolysis, but the half-lives of these cross-links can be as long as several hours. Such long-lived DPCs are extremely toxic due to their large sizes, which physically block DNA replication. Therefore, these adducts must be promptly eradicated to maintain genome integrity. Herein, we used in vitro reconstitution experiments with chemically synthesized, stable, and site-specific Schiff base AP-peptide/protein cross-link analogs to demonstrate for the first time that this type of DPC can be repaired by Escherichia coli (E. coli) long-patch base excision repair. We demonstrated that the repair process requires a minimum of three enzymes and five consecutive steps, including: (1) 5'-DNA strand incision of the DPC by endonuclease IV; (2 to 4) strand-displacement DNA synthesis, removal of the 5'-deoxyribose phosphate-peptide/protein adduct-containing flap, and gap-filling DNA synthesis by DNA polymerase I; and (5) strand ligation by a ligase. We further demonstrated that endonuclease IV plays a major role in incising an AP-peptide cross-link within E. coli cell extracts. We also report that eradicating model AP-protein (11.2-36.1 kDa) DPCs is less efficient than that of an AP-peptide 10mer cross-link, supporting the emerging model that proteolysis is likely required for efficient DPC repair., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

NAD + is a versatile biomolecule acting as a master regulator and substrate in various cellular processes, including redox regulation, metabolism, and various signaling pathways. In this article, we concisely and critically review the role of NAD + in mechanisms promoting genome maintenance. Numerous NAD + -dependent reactions are involved in the preservation of genome stability, the cellular DNA damage response, and other pathways regulating nucleic acid metabolism, such as gene expression and cell proliferation pathways. Of note, NAD + serves as a substrate to ADP-ribosyltransferases, sirtuins, and potentially also eukaryotic DNA ligases, all of which regulate various aspects of DNA integrity, damage repair, and gene expression. Finally, we critically analyze recent developments in the field as well as discuss challenges associated with therapeutic actions intended to raise NAD + levels., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

G-quadruplex (G4)-prone structures are abundant in mammalian genomes, where they have been shown to influence DNA replication, transcription, and genome stability. In this article, we constructed cells with a single ectopic homopurine/homopyrimidine repeat tract derived from the polycystic kidney disease type 1 (PKD1) locus, which is capable of forming triplex (H3) and G4 DNA structures. We show that ligand stabilization of these G4 structures results in deletions of the G4 consensus sequence, as well as kilobase deletions spanning the G4 and ectopic sites. Furthermore, we show that DNA double-strand breaks at the ectopic site are dependent on the nuclease Mus81. Hypermutagenesis during sister chromatid repair extends several kilobases from the G4 site and breaks at the G4 site resulting in microhomology-mediated translocations. To determine whether H3 or G4 structures are responsible for homopurine/homopyrimidine tract instability, we derived constructs and cell lines from the PKD1 repeat, which can only form H3 or G4 structures. Under normal growth conditions, we found that G4 cell lines lost the G4 consensus sequence early during clonal outgrowth, whereas H3 cells showed DNA instability early during outgrowth but only lost reporter gene expression after prolonged growth. Thus, both the H3 and G4 non-B conformation DNAs exhibit genomic instability, but they respond differently to endogenous replication stress. Our results show that the outcomes of replication-dependent double-strand breaks at non-B-DNAs model the instability observed in microhomology-mediated break-induced replication (BIR). Marked variability in the frequency of mutagenesis during BIR suggests possible dynamic heterogeneity in the BIR replisome., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Faithful replication of genomic DNA by high-fidelity DNA polymerases is crucial for the survival of most living organisms. While high-fidelity DNA polymerases favor canonical base pairs over mismatches by a factor of ∼1 × 10 5 , fidelity is further enhanced several orders of magnitude by a 3'-5' proofreading exonuclease that selectively removes mispaired bases in the primer strand. Despite the importance of proofreading to maintaining genome stability, it remains much less studied than the fidelity mechanisms employed at the polymerase active site. Here we characterize the substrate specificity for the proofreading exonuclease of a high-fidelity DNA polymerase by investigating the proofreading kinetics on various DNA substrates. The contribution of the exonuclease to net fidelity is a function of the kinetic partitioning between extension and excision. We show that while proofreading of a terminal mismatch is efficient, proofreading a mismatch buried by one or two correct bases is even more efficient. Because the polymerase stalls after incorporation of a mismatch and after incorporation of one or two correct bases on top of a mismatch, the net contribution of the exonuclease is a function of multiple opportunities to correct mistakes. We also characterize the exonuclease stereospecificity using phosphorothioate-modified DNA, provide a homology model for the DNA primer strand in the exonuclease active site, and propose a dynamic structural model for the transfer of DNA from the polymerase to the exonuclease active site based on MD simulations., Competing Interests: Conflict of interest K. A. J. is president of KinTek Corporation, which provided the RQF-3 rapid quench flow instrument and KinTek Explorer software used in this study., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Alpha-synuclein (aSyn) is a vertebrate protein, normally found within the presynaptic nerve terminal and nucleus, which is known to form somatic and neuritic aggregates in certain neurodegenerative diseases. Disease-associated aggregates of aSyn are heavily phosphorylated at serine-129 (pSyn), while normal aSyn protein is not. Within the nucleus, aSyn can directly bind DNA, but the mechanism of binding and the potential modulatory roles of phosphorylation are poorly understood. Here we demonstrate using a combination of electrophoretic mobility shift assay and atomic force microscopy approaches that both aSyn and pSyn can bind DNA within the major groove, in a DNA length-dependent manner and with little specificity for DNA sequence. Our data are consistent with a model in which multiple aSyn molecules bind a single 300 base pair (bp) DNA molecule in such a way that stabilizes the DNA in a bent conformation. We propose that serine-129 phosphorylation decreases the ability of aSyn to both bind and bend DNA, as aSyn binds 304 bp circular DNA forced into a bent shape, but pSyn does not. Two aSyn paralogs, beta- and gamma-synuclein, also interact with DNA differently than aSyn, and do not stabilize similar DNA conformations. Our work suggests that reductions in aSyn's ability to bind and bend DNA induced by serine-129 phosphorylation may be important for modulating aSyn's known roles in DNA metabolism, including the regulation of transcription and DNA repair., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Bromodomain adjacent to zinc finger domain protein 2A (BAZ2A) (also called transcription termination factor-1 interacting protein 5), a key component of the nucleolar remodeling complex, recruits the nucleolar remodeling complex to ribosomal RNA genes, leading to their transcriptional repression. In addition to its tandem plant homeodomain-bromodomain that is involved in binding to acetylated histone H4, BAZ2A also contains a methyl-CpG-binding domain (MBD)-like Tip5/ARBP/MBD (TAM) domain that shares sequence homology with the MBD. In contrast with the methyl-CpG-binding ability of the canonical MBD, the BAZ2A TAM domain has been shown to bind to promoter-associated RNAs of ribosomal RNA genes and promoter DNAs of other genes independent of DNA methylation. Nevertheless, how the TAM domain binds to RNA/DNA mechanistically remains elusive. Here, we characterized the DNA-/RNA-binding basis of the BAZ2A TAM domain by EMSAs, isothermal titration calorimetry binding assays, mutagenesis analysis, and X-ray crystallography. Our results showed that the TAM domain of BAZ2A selectively binds to dsDNA and dsRNA and that it binds to the backbone of dsDNA in a sequence nonspecific manner, which is distinct from the base-specific binding of the canonical MBD. Thus, our results explain why the TAM domain of BAZ2A does not specifically bind to mCG or TG dsDNA like the canonical MBD and also provide insights for further biological study of BAZ2A acting as a transcription factor in the future., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

DNA-protein cross-links are formed when proteins become covalently trapped with DNA in the presence of exogenous or endogenous alkylating agents. If left unrepaired, they inhibit transcription as well as DNA unwinding during replication and may result in genome instability or even cell death. The DNA repair protein O 6 -alkylguanine DNA-alkyltransferase (AGT) is known to form DNA cross-links in the presence of the carcinogen 1,2-dibromoethane, resulting in G:C to T:A transversions and other mutations in both bacterial and mammalian cells. We hypothesized that AGT-DNA cross-links would be processed by nuclear proteases to yield peptides small enough to be bypassed by translesion (TLS) polymerases. Here, a 15-mer and a 36-mer peptide from the active site of AGT were cross-linked to the N2 position of guanine via conjugate addition of a thiol containing a peptide dehydroalanine moiety. Bypass studies with DNA polymerases (pols) η and κ indicated that both can accurately bypass the cross-linked DNA peptides. The specificity constant (k cat /K m ) for steady-state incorporation of the correct nucleotide dCTP increased by 6-fold with human (h) pol κ and 3-fold with hpol η, with hpol η preferentially inserting nucleotides in the order dC > dG > dA > dT. LC-MS/MS analysis of the extension product also revealed error-free bypass of the cross-linked 15-mer peptide by hpol η. We conclude that a bulky 15-mer AGT peptide cross-linked to the N2 position of guanine can retard polymerization, but that overall fidelity is not compromised because only correct bases are inserted and extended., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

RAD51-associated protein 1 (RAD51AP1) is a key protein in the homologous recombination (HR) DNA repair pathway. Loss of RAD51AP1 leads to defective HR, genome instability, and telomere erosion. RAD51AP1 physically interacts with the RAD51 recombinase and promotes RAD51-mediated capture of donor DNA, synaptic complex assembly, and displacement-loop formation when tested with nucleosome-free DNA substrates. In cells, however, DNA is packaged into chromatin, posing an additional barrier to the complexities of the HR reaction. In this study, we show that RAD51AP1 binds to nucleosome core particles (NCPs), the minimum basic unit of chromatin in which approximately two superhelical turns of 147 bp double-stranded DNA are wrapped around one histone octamer with no free DNA ends remaining. We identified a C-terminal region in RAD51AP1, including its previously mapped DNA-binding domain, as critical for mediating the association between RAD51AP1 and both the NCP and the histone octamer. Using in vitro surrogate assays of HR activity, we show that RAD51AP1 is capable of promoting duplex DNA capture and initiating joint-molecule formation with the NCP and chromatinized template DNA, respectively. Together, our results suggest that RAD51AP1 directly assists in the RAD51-mediated search for donor DNA in chromatin. We present a model, in which RAD51AP1 anchors the DNA template through affinity for its nucleosomes to the RAD51-ssDNA nucleoprotein filament., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the content of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The determination of the double helical structure of DNA in 1953 remains the landmark event in the development of modern biological and biomedical science. This structure has also been the starting point for the determination of some 2000 DNA crystal structures in the subsequent 68 years. Their structural diversity has extended to the demonstration of sequence-dependent local structure in duplex DNA, to DNA bending in short and long sequences and in the DNA wound round the nucleosome, and to left-handed duplex DNAs. Beyond the double helix itself, in circumstances where DNA sequences are or can be induced to unwind from being duplex, a wide variety of topologies and forms can exist. Quadruplex structures, based on four-stranded cores of stacked G-quartets, are prevalent though not randomly distributed in the human and other genomes and can play roles in transcription, translation, and replication. Yet more complex folds can result in DNAs with extended tertiary structures and enzymatic/catalytic activity. The Protein Data Bank is the depository of all these structures, and the resource where structures can be critically examined and validated, as well as compared one with another to facilitate analysis of conformational and base morphology features. This review will briefly survey the major structural classes of DNAs and illustrate their significance, together with some examples of how the use of the Protein Data Bank by for example, data mining, has illuminated DNA structural concepts., Competing Interests: Conflict of interest The author declares that he has no conflicts of interest with the contents of this manuscript., (Copyright © 2021 The Author. Published by Elsevier Inc. All rights reserved.)

My personal and professional journeys have been far from predictable based on my early childhood. Owing to a range of serendipitous influences, I miraculously transitioned from a rebellious, apathetic teenage street urchin who did poorly in school to a highly motivated, disciplined, and ambitious academic honors student. I was the proverbial "late bloomer." Ultimately, I earned my PhD in biophysical chemistry at Yale, followed by a postdoc fellowship at Berkeley. These two meccas of thermodynamics, coupled with my deep fascination with biology, instilled in me a passion to pursue an academic career focused on mapping the energy landscapes of biological systems. I viewed differential energetics as the language of molecular communication that would dictate and control biological structures, as well as modulate the modes of action associated with biological functions. I wanted to be a "molecular linguist." For the next 50 years, my group and I used a combination of spectroscopic and calorimetric techniques to characterize the energy profiles of the polymorphic conformational space of DNA molecules, their differential ligand-binding properties, and the energy landscapes associated with mutagenic DNA damage recognition, repair, and replication. As elaborated below, the resultant energy databases have enabled the development of quantitative molecular biology through the rational design of primers, probes, and arrays for diagnostic, therapeutic, and molecular-profiling protocols, which collectively have contributed to a myriad of biomedical assays. Such profiling is further justified by yielding unique energy-based insights that complement and expand elegant, structure-based understandings of biological processes., Competing Interests: Conflict of interest The author declares that he has no conflicts of interest with the contents of this article., (Copyright © 2021 The Author. Published by Elsevier Inc. All rights reserved.)

Unrepaired DNA-protein cross-links, due to their bulky nature, can stall replication forks and result in genome instability. Large DNA-protein cross-links can be cleaved into DNA-peptide cross-links, but the extent to which these smaller fragments disrupt normal replication is not clear. Ethylene dibromide (1,2-dibromoethane) is a known carcinogen that can cross-link the repair protein O 6 -alkylguanine-DNA alkyltransferase (AGT) to the N6 position of deoxyadenosine (dA) in DNA, as well as four other positions in DNA. We investigated the effect of a 15-mer peptide from the active site of AGT, cross-linked to the N6 position of dA, on DNA replication by human translesion synthesis DNA polymerases (Pols) η, ⍳, and κ. The peptide-DNA cross-link was bypassed by the three polymerases at different rates. In steady-state kinetics, the specificity constant (k cat /K m ) for incorporation of the correct nucleotide opposite to the adduct decreased by 220-fold with Pol κ, tenfold with pol η, and not at all with Pol ⍳. Pol η incorporated all four nucleotides across from the lesion, with the preference dT > dC > dA > dG, while Pol ⍳ and κ only incorporated the correct nucleotide. However, LC-MS/MS analysis of the primer-template extension product revealed error-free bypass of the cross-linked 15-mer peptide by Pol η. We conclude that a bulky 15-mer peptide cross-linked to the N6 position of dA can retard polymerization and cause miscoding but that overall fidelity is not compromised because only correct pairs are extended., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Methods for rapid and high-throughput screening of transcription in vitro to examine reaction conditions, enzyme mutants, promoter variants, and small molecule modulators can be extremely valuable tools. However, these techniques may be difficult to establish or inaccessible to many researchers. To develop a straightforward and cost-effective platform for assessing transcription in vitro, we used the "Broccoli" RNA aptamer as a direct, real-time fluorescent transcript readout. To demonstrate the utility of our approach, we screened the effect of common reaction conditions and components on bacteriophage T7 RNA polymerase (RNAP) activity using a common quantitative PCR instrument for fluorescence detection. Several essential conditions for in vitro transcription by T7 RNAP were confirmed with this assay, including the importance of enzyme and substrate concentrations, covariation of magnesium and nucleoside triphosphates, and the effects of several typical additives. When we used this method to assess all possible point mutants of a canonical T7 RNAP promoter, our results coincided well with previous reports. This approach should translate well to a broad variety of bacteriophage in vitro transcription systems and provides a platform for developing fluorescence-based readouts of more complex transcription systems in vitro., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

All cellular genetic information is transcribed into RNA by multisubunit RNA polymerases (RNAPs). The basal transcription initiation factors of cellular RNAPs stimulate the initial RNA synthesis via poorly understood mechanisms. Here, we explored the mechanism employed by the bacterial factor σ in promoter-independent initial transcription. We found that the RNAP holoenzyme lacking the promoter-binding domain σ4 is ineffective in de novo transcription initiation and displays high propensity to pausing upon extension of RNAs 3 to 7 nucleotides in length. The nucleotide at the RNA 3' end determines the pause lifetime. The σ4 domain stabilizes short RNA:DNA hybrids and suppresses pausing by stimulating RNAP active-center translocation. The antipausing activity of σ4 is modulated by its interaction with the β subunit flap domain and by the σ remodeling factors AsiA and RbpA. Our results suggest that the presence of σ4 within the RNA exit channel compensates for the intrinsic instability of short RNA:DNA hybrids by increasing RNAP processivity, thus favoring productive transcription initiation. This "RNAP boosting" activity of the initiation factor is shaped by the thermodynamics of RNA:DNA interactions and thus, should be relevant for any factor-dependent RNAP., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Ribonuclease HI, an endoribonuclease, catalyzes the hydrolysis of the RNA strand of an RNA/DNA hybrid and requires divalent metal ions for its enzymatic activity. However, the mechanistic details of the activity of ribonuclease HI and its interaction with divalent metal ions remain unclear. In this study, we performed real-time monitoring of the enzyme-substrate complex in the presence of divalent metal ions (Mn 2+ or Zn 2+ ) using electrospray ionization-mass spectrometry (ESI-MS). The findings provide clear evidence that the enzymatic activity of the ternary complex requires the binding of two divalent metal ions. The Zn 2+ ions bind to both the enzyme itself and the enzyme:substrate complex more strongly than Mn 2+ ions, and gives, in part, the ternary complex, [RNase HI:nicked RNA/DNA hybrid:2Zn 2+ ], suggesting that the ternary complex is retained, even after the hydrolysis of the substrate. The collective results presented herein shed new light on the essential role of divalent metal ions in the activity of ribonuclease HI and demonstrate how Zn 2+ ions confer inhibitory properties on the activity of this enzyme by forming a highly stable complex with the substrate., Competing Interests: Conflict of interest The authors declare no conflicts of interest in regard to this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Bacterial low-copy-number plasmids require partition (par) systems to ensure their stable inheritance by daughter cells. In general, these systems consist of three components: a centromeric DNA sequence, a centromere-binding protein and a nucleotide hydrolase that polymerizes and functions as a motor. Type III systems, however, segregate plasmids using three proteins: the FtsZ/tubulin-like GTPase TubZ, the centromere-binding protein TubR and the MerR-like transcriptional regulator TubY. Although the TubZ filament is sufficient to transport the TubR-centromere complex in vitro, TubY is still necessary for the stable maintenance of the plasmid. TubY contains an N-terminal DNA-binding helix-turn-helix motif and a C-terminal coiled-coil followed by a cluster of lysine residues. This study determined the crystal structure of the C-terminal domain of TubY from the Bacillus cereus pXO1-like plasmid and showed that it forms a tetrameric parallel four-helix bundle that differs from the typical MerR family proteins with a dimeric anti-parallel coiled-coil. Biochemical analyses revealed that the C-terminal tail with the conserved lysine cluster helps TubY to stably associate with the TubR-centromere complex as well as to nonspecifically bind DNA. Furthermore, this C-terminal tail forms an amphipathic helix in the presence of lipids but must oligomerize to localize the protein to the membrane in vivo. Taken together, these data suggest that TubY is a component of the nucleoprotein complex within the partitioning machinery, and that lipid membranes act as mediators of type III systems., (Copyright © 2020 © 2020 Hayashi. Published by Elsevier Inc. All rights reserved.)

Investigations of bacterial resistance strategies can aid in the development of new antimicrobial drugs as a countermeasure to the increasing worldwide prevalence of bacterial antibiotic resistance. One such strategy involves the TipA class of transcription factors, which constitute minimal autoregulated multidrug resistance (MDR) systems against diverse antibiotics. However, we have insufficient information regarding how antibiotic binding induces transcriptional activation to design molecules that could interfere with this process. To learn more, we determined the crystal structure of SkgA from Caulobacter crescentus as a representative TipA protein. We identified an unexpected spatial orientation and location of the antibiotic-binding TipAS effector domain in the apo state. We observed that the α6-α7 region of the TipAS domain, which is canonically responsible for forming the lid of antibiotic-binding cleft to tightly enclose the bound antibiotic, is involved in the dimeric interface and stabilized via interaction with the DNA-binding domain in the apo state. Further structural and biochemical analyses demonstrated that the unliganded TipAS domain sterically hinders promoter DNA binding but undergoes a remarkable conformational shift upon antibiotic binding to release this autoinhibition via a switch of its α6-α7 region. Hence, the promoters for MDR genes including tipA and RNA polymerases become available for transcription, enabling efficient antibiotic resistance. These insights into the molecular mechanism of activation of TipA proteins advance our understanding of TipA proteins, as well as bacterial MDR systems, and may provide important clues to block bacterial resistance., (Copyright © 2020 © 2020 Jiang et al. Published by Elsevier Inc. All rights reserved.)

DNA polymerase from bacteriophage T7 undergoes large, substrate-induced conformational changes that are thought to account for high replication fidelity, but prior studies were adversely affected by mutations required to construct a Cys-lite variant needed for site-specific fluorescence labeling. Here we have optimized the direct incorporation of a fluorescent un-natural amino acid, (7-hydroxy-4-coumarin-yl)-ethylglycine, using orthogonal amber suppression machinery in Escherichia coli MS methods verify that the unnatural amino acid is only incorporated at one position with minimal background. We show that the single fluorophore provides a signal to detect nucleotide-induced conformational changes through equilibrium and stopped-flow kinetic measurements of correct nucleotide binding and incorporation. Pre-steady-state chemical quench methods show that the kinetics and fidelity of DNA replication catalyzed by the labeled enzyme are largely unaffected by the unnatural amino acid. These advances enable rigorous analysis to establish the kinetic and mechanistic basis for high-fidelity DNA replication., Competing Interests: Conflict of interest—K. A. J. is president of KinTek Corporation, which provided the SF300x stopped-flow and RQF-3 rapid quench-flow instruments and KinTek Explorer software used in this study., (© 2020 Dangerfield and Johnson.)

In eukaryotic DNA replication, DNA polymerase ε (Polε) is responsible for leading strand synthesis, whereas DNA polymerases α and δ synthesize the lagging strand. The human Polε (hPolε) holoenzyme is comprised of the catalytic p261 subunit and the noncatalytic p59, p17, and p12 small subunits. So far, the contribution of the noncatalytic subunits to hPolε function is not well understood. Using pre-steady-state kinetic methods, we established a minimal kinetic mechanism for DNA polymerization and editing catalyzed by the hPolε holoenzyme. Compared with the 140-kDa N-terminal catalytic fragment of p261 (p261N), which we kinetically characterized in our earlier studies, the presence of the p261 C-terminal domain (p261C) and the three small subunits increased the DNA binding affinity and the base substitution fidelity. Although the small subunits enhanced correct nucleotide incorporation efficiency, there was a wide range of rate constants when incorporating a correct nucleotide over a single-base mismatch. Surprisingly, the 3'→5' exonuclease activity of the hPolε holoenzyme was significantly slower than that of p261N when editing both matched and mismatched DNA substrates. This suggests that the presence of p261C and the three small subunits regulates the 3'→5' exonuclease activity of the hPolε holoenzyme. Together, the 3'→5' exonuclease activity and the variable mismatch extension activity modulate the overall fidelity of the hPolε holoenzyme by up to 3 orders of magnitude. Thus, the presence of p261C and the three noncatalytic subunits optimizes the dual enzymatic activities of the catalytic p261 subunit and makes the hPolε holoenzyme an efficient and faithful replicative DNA polymerase., Competing Interests: Conflict of interest—The authors declare no conflicts of interest in regards to this manuscript., (© 2020 Zahurancik and Suo.)

As the Watson-Crick faces of nucleobases are protected in dsDNA, it is commonly assumed that deleterious alkylation damage to the Watson-Crick faces of nucleobases predominantly occurs when DNA becomes single-stranded during replication and transcription. However, damage to the Watson-Crick faces of nucleobases has been reported in dsDNA in vitro through mechanisms that are not understood. In addition, the extent of protection from methylation damage conferred by dsDNA relative to ssDNA has not been quantified. Watson-Crick base pairs in dsDNA exist in dynamic equilibrium with Hoogsteen base pairs that expose the Watson-Crick faces of purine nucleobases to solvent. Whether this can influence the damage susceptibility of dsDNA remains unknown. Using dot-blot and primer extension assays, we measured the susceptibility of adenine-N1 to methylation by dimethyl sulfate (DMS) when in an A-T Watson-Crick versus Hoogsteen conformation. Relative to unpaired adenines in a bulge, Watson-Crick A-T base pairs in dsDNA only conferred ∼130-fold protection against adenine-N1 methylation, and this protection was reduced to ∼40-fold for A( syn )-T Hoogsteen base pairs embedded in a DNA-drug complex. Our results indicate that Watson-Crick faces of nucleobases are accessible to alkylating agents in canonical dsDNA and that Hoogsteen base pairs increase this accessibility. Given the higher abundance of dsDNA relative to ssDNA, these results suggest that dsDNA could be a substantial source of cytotoxic damage. The work establishes DMS probing as a method for characterizing A( syn )-T Hoogsteen base pairs in vitro and also lays the foundation for a sequencing approach to map A( syn )-T Hoogsteen and unpaired adenines genome-wide in vivo., Competing Interests: Conflict of interest—The authors declare that they have no conflict of interest with the contents of this article., (© 2020 Xu et al.)

Among the multiple antiviral defense mechanisms found in prokaryotes, CRISPR-Cas systems stand out as the only known RNA-programmed pathways for detecting and destroying bacteriophages and plasmids. Class 1 CRISPR-Cas systems, the most widespread and diverse of these adaptive immune systems, use an RNA-guided multiprotein complex to find foreign nucleic acids and trigger their destruction. In this review, we describe how these multisubunit complexes target and cleave DNA and RNA and how regulatory molecules control their activities. We also highlight similarities to and differences from Class 2 CRISPR-Cas systems, which use a single-protein effector, as well as other types of bacterial and eukaryotic immune systems. We summarize current applications of the Class 1 CRISPR-Cas systems for DNA/RNA modification, control of gene expression, and nucleic acid detection., Competing Interests: Conflict of interest—The Regents of the University of California have patents issued for CRISPR technologies on which J. A. D. is an inventor and patents pending for CRISPR technologies on which both J. A. D. and T. Y. L. are inventors. J. A. D. is a co-founder of Caribou Biosciences, Editas Medicine, Intellia Therapeutics, Scribe Therapeutics, and Mammoth Biosciences. J. A. D. is a scientific advisor to Caribou Biosciences, Intellia Therapeutics, eFFECTOR Therapeutics, Scribe Therapeutics, Synthego, Algen Biotechnologies, Felix Biosciences, Mammoth Biosciences, and Inari. J. A. D. is a Director at Johnson & Johnson and has research projects sponsored by Pfizer, Roche, AppleTree Partners, and Biogen.

Replication protein A (RPA), a major eukaryotic ssDNA-binding protein, is essential for all metabolic processes that involve ssDNA, including DNA replication, repair, and damage signaling. To perform its functions, RPA binds ssDNA tightly. In contrast, it was presumed that RPA binds RNA weakly. However, recent data suggest that RPA may play a role in RNA metabolism. RPA stimulates RNA-templated DNA repair in vitro and associates in vivo with R-loops, the three-stranded structures consisting of an RNA-DNA hybrid and the displaced ssDNA strand. R-loops are common in the genomes of pro- and eukaryotes, including humans, and may play an important role in transcription-coupled homologous recombination and DNA replication restart. However, the mechanism of R-loop formation remains unknown. Here, we investigated the RNA-binding properties of human RPA and its possible role in R-loop formation. Using gel-retardation and RNA/DNA competition assays, we found that RPA binds RNA with an unexpectedly high affinity ( K D ≈ 100 pm). Furthermore, RPA, by forming a complex with RNA, can promote R-loop formation with homologous dsDNA. In reconstitution experiments, we showed that human DNA polymerases can utilize RPA-generated R-loops for initiation of DNA synthesis, mimicking the process of replication restart in vivo These results demonstrate that RPA binds RNA with high affinity, supporting the role of this protein in RNA metabolism and suggesting a mechanism of genome maintenance that depends on RPA-mediated DNA replication restart., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Mazina et al.)

DNA polymerase (pol) β catalyzes two reactions at DNA gaps generated during base excision repair, gap-filling DNA synthesis and lyase-dependent 5´-end deoxyribose phosphate removal. The lyase domain of pol β has been proposed to function in DNA gap recognition and to facilitate DNA scanning during substrate search. However, the mechanisms and molecular interactions used by pol β for substrate search and recognition are not clear. To provide insight into this process, a comparison was made of the DNA binding affinities of WT pol β, pol λ, and pol μ, and several variants of pol β, for 1-nt-gap-containing and undamaged DNA. Surprisingly, this analysis revealed that mutation of three lysine residues in the lyase active site of pol β, 35, 68, and 72, to alanine (pol β KΔ3A) increased the binding affinity for nonspecific DNA ∼11-fold compared with that of the WT. WT pol μ, lacking homologous lysines, displayed nonspecific DNA binding behavior similar to that of pol β KΔ3A, in line with previous data demonstrating both enzymes were deficient in processive searching. In fluorescent microscopy experiments using mouse fibroblasts deficient in PARP-1, the ability of pol β KΔ3A to localize to sites of laser-induced DNA damage was strongly decreased compared with that of WT pol β. These data suggest that the three lysines in the lyase active site destabilize pol β when bound to DNA nonspecifically, promoting DNA scanning and providing binding specificity for gapped DNA., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

AlkB is a bacterial Fe(II)- and 2-oxoglutarate-dependent dioxygenase that repairs a wide range of alkylated nucleobases in DNA and RNA as part of the adaptive response to exogenous nucleic acid-alkylating agents. Although there has been longstanding interest in the structure and specificity of Escherichia coli AlkB and its homologs, difficulties in assaying their repair activities have limited our understanding of their substrate specificities and kinetic mechanisms. Here, we used quantitative kinetic approaches to determine the transient kinetics of recognition and repair of alkylated DNA by AlkB. These experiments revealed that AlkB is a much faster alkylation repair enzyme than previously reported and that it is significantly faster than DNA repair glycosylases that recognize and excise some of the same base lesions. We observed that whereas 1, N 6 -ethenoadenine can be repaired by AlkB with similar efficiencies in both single- and double-stranded DNA, 1-methyladenine is preferentially repaired in single-stranded DNA. Our results lay the groundwork for future studies of AlkB and its human homologs ALKBH2 and ALKBH3., (© 2020 Baldwin et al.)

Site-specific arrest of RNA polymerases (RNAPs) is fundamental to several technologies that assess RNA structure and function. Current in vitro transcription "roadblocking" approaches inhibit transcription elongation by blocking RNAP with a protein bound to the DNA template. One limitation of protein-mediated transcription roadblocking is that it requires inclusion of a protein factor extrinsic to the minimal in vitro transcription reaction. In this work, we developed a chemical approach for halting transcription by Escherichia coli RNAP. We first established a sequence-independent method for site-specific incorporation of chemical lesions into dsDNA templates by sequential PCR and translesion synthesis. We then show that interrupting the transcribed DNA strand with an internal desthiobiotin-triethylene glycol modification or 1,N 6 -etheno-2'-deoxyadenosine base efficiently and stably halts Escherichia coli RNAP transcription. By encoding an intrinsic stall site within the template DNA, our chemical transcription roadblocking approach enables display of nascent RNA molecules from RNAP in a minimal in vitro transcription reaction., (© 2020 Strobel et al.)

Forkhead box N1 (FOXN1) is a member of the forkhead box family of transcription factors and plays an important role in thymic epithelial cell differentiation and development. FOXN1 mutations in humans and mice give rise to the "nude" phenotype, which is marked by athymia. FOXN1 belongs to a subset of the FOX family that recognizes an alternative forkhead-like (FHL) consensus sequence (GACGC) that is different from the more widely recognized forkhead (FKH) sequence RYAAAYA (where R is purine, and Y is pyrimidine). Here, we present the FOXN1 structure in complex with DNA containing an FHL motif at 1.6 Å resolution, in which the DNA sequence is recognized by a mixture of direct and water-mediated contacts provided by residues in an α-helix inserted in the DNA major groove (the recognition helix). Comparisons with the structure of other FOX family members revealed that the FKH and FHL DNA sequences are bound in two distinct modes, with partially different registers for the protein DNA contacts. We identified a single alternative rotamer within the recognition helix itself as an important determinant of DNA specificity and found protein sequence features in the recognition helix that could be used to predict the specificity of other FOX family members. Finally, we demonstrate that the C-terminal region of FOXN1 is required for high-affinity DNA binding and that FOXN1 has a significantly reduced affinity for DNA that contains 5'-methylcytosine, which may have implications for the role of FOXN1 in thymic involution., (© 2020 Newman et al.)

Quorum sensing is a bacterial communication process whereby bacteria produce, release, and detect extracellular signaling molecules called autoinducers to coordinate collective behaviors. In the pathogen Vibrio cholerae , the quorum-sensing autoinducer 3,5-dimethyl-pyrazin-2-ol (DPO) binds the receptor and transcription factor VqmA. The DPO-VqmA complex activates transcription of vqmR , encoding the VqmR small RNA, which represses genes required for biofilm formation and virulence factor production. Here, we show that VqmA is soluble and properly folded and activates basal-level transcription of its target vqmR in the absence of DPO. VqmA transcriptional activity is increased in response to increasing concentrations of DPO, allowing VqmA to drive the V. cholerae quorum-sensing transition at high cell densities. We solved the DPO-VqmA crystal structure to 2.0 Å resolution and compared it with existing structures to understand the conformational changes VqmA undergoes upon DNA binding. Analysis of DPO analogs showed that a hydroxyl or carbonyl group at the 2'-position is critical for binding to VqmA. The proposed DPO precursor, a linear molecule, N -alanyl-aminoacetone (Ala-AA), also bound and activated VqmA. Results from site-directed mutagenesis and competitive ligand-binding analyses revealed that DPO and Ala-AA occupy the same binding site. In summary, our structure-function analysis identifies key features required for VqmA activation and DNA binding and establishes that, whereas VqmA binds two different ligands, VqmA does not require a bound ligand for folding or basal transcriptional activity. However, bound ligand is required for maximal activity., (© 2020 Huang et al.)