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Burkholderia cenocepacia is an opportunistic and infective bacterium containing an orphan DNA methyltransferase called M.BceJIV with roles in regulating gene expression and motility of the bacterium. M.BceJIV recognizes a GTWWAC motif (where W can be an adenine or a thymine) and methylates N6 of the adenine at the fifth base position. Here, we present crystal structures of M.BceJIV/DNA/sinefungin ternary complex and allied biochemical, computational, and thermodynamic analyses. Remarkably, the structures show not one, but two DNA substrates bound to the M.BceJIV dimer, with each monomer contributing to the recognition of two recognition sequences. We also show that methylation at the two recognition sequences occurs independently, and that the GTWWAC motifs are enriched in intergenic regions in the genomes of B. cenocepacia strains. We further computationally assess the interactions underlying the affinities of different ligands (SAM, SAH, and sinefungin) for M.BceJIV, as a step towards developing selective inhibitors for limiting B. cenocepacia infection. Crystal structures of DNA methyltransferase M.BceJIV in complex with DNA and sinefungin reveal an unusual mode of DNA binding and methylation, wherein each M.BceJIV monomer contributes to the recognition of two DNA sequences. [ABSTRACT FROM AUTHOR]

A short prokaryotic Argonaute (pAgo) TIR-APAZ (SPARTA) defense system, activated by invading DNA to unleash its TIR domain for NAD(P)+ hydrolysis, was recently identified in bacteria. We report the crystal structure of SPARTA heterodimer in the absence of guide-RNA/target-ssDNA (2.66 Å) and a cryo-EM structure of the SPARTA oligomer (tetramer of heterodimers) bound to guide-RNA/target-ssDNA at nominal 3.15–3.35 Å resolution. The crystal structure provides a high-resolution view of SPARTA, revealing the APAZ domain as equivalent to the N, L1, and L2 regions of long pAgos and the MID domain containing a unique insertion (insert57). Cryo-EM structure reveals regions of the PIWI (loop10-9) and APAZ (helix αN) domains that reconfigure for nucleic-acid binding and decrypts regions/residues that reorganize to expose a positively charged pocket for higher-order assembly. The TIR domains amass in a parallel-strands arrangement for catalysis. We visualize SPARTA before and after RNA/ssDNA binding and uncover the basis of its active assembly leading to abortive infection. The SPARTA defense system, activated by invading DNA for NAD(P)+ hydrolysis, was recently identified in bacteria. Here, authors visualize SPARTA before and after nucleic acid binding and uncover the basis of its active assembly leading to abortive infection. [ABSTRACT FROM AUTHOR]

The B-family multi-subunit DNA polymerase ζ (Polζ) is important for translesion DNA synthesis (TLS) during replication, due to its ability to extend synthesis past nucleotides opposite DNA lesions and mismatched base pairs. We present a cryo-EM structure of Saccharomyces cerevisiae Polζ with an A:C mismatch at the primer terminus. The structure shows how the Polζ active site responds to the mismatched duplex DNA distortion, including the loosening of key protein-DNA interactions and a fingers domain in an "open" conformation, while the incoming dCTP is still able to bind for the extension reaction. The structure of the mismatched DNA-Polζ ternary complex reveals insights into mechanisms that either stall or favor continued DNA synthesis in eukaryotes. The structure of mismatched DNA-Polζ ternary complex provides a basis for understanding what makes Polζ adept at extending DNA synthesis past mismatched base pairs. [ABSTRACT FROM AUTHOR]

Triple negative breast cancer (TNBC) remains challenging because of heterogeneous responses to chemotherapy. Incomplete response is associated with a greater risk of metastatic progression. Therefore, treatments that target chemotherapy-resistant TNBC and enhance chemosensitivity would improve outcomes for these high-risk patients. Breast cancer stem cell-like cells (BCSCs) have been proposed to represent a chemotherapy-resistant subpopulation responsible for tumor initiation, progression and metastases. Targeting this population could lead to improved TNBC disease control. Here, we describe a novel multi-kinase inhibitor, 108600, that targets the TNBC BCSC population. 108600 treatment suppresses growth, colony and mammosphere forming capacity of BCSCs and induces G2M arrest and apoptosis of TNBC cells. In vivo, 108600 treatment of mice bearing triple negative tumors results in the induction of apoptosis and overcomes chemotherapy resistance. Finally, treatment with 108600 and chemotherapy suppresses growth of pre-established TNBC metastases, providing additional support for the clinical translation of this agent to clinical trials. Finding therapeutic strategies for aggressive triple negative breast cancer (TNBC) is an important challenge in clinical practice. Here, the authors identify a multi-kinases inhibitor with antitumor activity and able overcome chemotherapy resistance of TNBC in vivo. [ABSTRACT FROM AUTHOR]

The size of the transcriptional program of long non-coding RNAs in the mammalian genome has engendered discussions about their biological roles1, particularly the promoter antisense (PAS) transcripts2,3. Here we report the development of an assay—referred to as chromatin isolation by RNA–Cas13a complex—to quantitatively detect the distribution of RNA in the genome. The assay revealed that PAS RNAs serve as a key gatekeeper of a broad transcriptional pause release program, based on decommissioning the 7SK small nuclear RNA-dependent inhibitory P-TEFb complex. Induction of PAS RNAs by liganded ERα led to a significant loss of H3K9me3 and the release of basally recruited HP1α and KAP1 on activated target gene promoters. This release was due to PAS RNA-dependent recruitment of H3K9me3 demethylases, which required interactions with a compact stem-loop structure in the PAS RNAs, an apparent feature of similarly regulated PAS RNAs. Activation of the ERα-bound MegaTrans enhancer, which is essential for robust pause release, required the recruitment of phosphorylated KAP1, with its transfer to the cognate promoters permitting 17β-oestradiol-induced pause release and activation of the target gene. This study reveals a mechanism, based on RNA structure, that mediates the function of PAS RNAs in gene regulation.The authors describe a role for the non-coding antisense transcripts produced at promoters in regulating ligand-induced activation of gene transcription. [ABSTRACT FROM AUTHOR]

PrimPol is a human DNA polymerase-primase that localizes to mitochondria and nucleus and bypasses the major oxidative lesion 7,8-dihydro-8-oxoguanine (oxoG) via translesion synthesis, in mostly error-free manner. We present structures of PrimPol insertion complexes with a DNA template-primer and correct dCTP or erroneous dATP opposite the lesion, as well as extension complexes with C or A as a 3′−terminal primer base. We show that during the insertion of C and extension from it, the active site is unperturbed, reflecting the readiness of PrimPol to accommodate oxoG(anti). The misinsertion of A opposite oxoG(syn) also does not alter the active site, and is likely less favorable due to lower thermodynamic stability of the oxoG(syn)•A base-pair. During the extension step, oxoG(syn) induces an opening of its base-pair with A or misalignment of the 3′-A primer terminus. Together, the structures show how PrimPol accurately synthesizes DNA opposite oxidatively damaged DNA in human cells. The human DNA primase and DNA polymerase PrimPol replicates through the major oxidative DNA damage lesion 7,8-dihydro-8-oxoguanine (oxoG) via translesion synthesis in a mostly error-free manner thus suppressing oxoG-induced mutagenesis in mitochondria and the nucleus. Here, the authors present crystal structures of PrimPol in complex with an oxoG lesion in different contexts that provide mechanistic insights into how PrimPol performs predominantly accurate synthesis on oxidative-damaged DNAs in human cells. [ABSTRACT FROM AUTHOR]

Benzo[a]pyrene (BP) is a carcinogen in cigarette smoke which, after metabolic activation, can react with the exocyclic N² amino group of guanine to generate four stereoisomeric BP-N²-dG adducts. Rev1 is unique among translesion synthesis DNA polymerases in employing a protein-template-directed mechanism of DNA synthesis opposite undamaged and damaged guanine. Here we report high-resolution structures of yeast Rev1 with three BP-N²-dG adducts, namely the 10S (+)-trans-BP-N²-dG, 10R (+)-cis-BP-N²-dG, and 10S ( - )-cis-BP-N²-dG. Surprisingly, in all three structures, the bulky and hydrophobic BP pyrenyl residue is entirely solvent-exposed in the major groove of the DNA. This is very different from the adduct alignments hitherto observed in free or protein-bound DNA. All complexes are well poised for dCTP insertion. Our structures provide a view of cis-BP-N²-dG adducts in a DNA polymerase active site, and offer a basis for understanding error-free replication of the BP-derived stereoisomeric guanine adducts. [ABSTRACT FROM AUTHOR]

A major clinical problem in the use of cisplatin to treat cancers is tumor resistance. DNA polymerase ? (Pol-?) is a crucial polymerase that allows cancer cells to cope with the cisplatin-DNA adducts that are formed during chemotherapy. We present here a structure of human Pol-? inserting deoxycytidine triphosphate (dCTP) opposite a cisplatin intrastrand cross-link (PtGpG). We show that the specificity of human Pol-? for PtGpG derives from an active site that is open to permit Watson-Crick geometry of the nascent PtGpG-dCTP base pair and to accommodate the lesion without steric hindrance. This specificity is augmented by the residues Gln38 and Ser62, which interact with PtGpG, and Arg61, which interacts with the incoming dCTP. Collectively, the structure provides a basis for understanding how Pol-? in human cells can tolerate the DNA damage caused by cisplatin chemotherapy and offers a framework for the design of inhibitors in cancer therapy. [ABSTRACT FROM AUTHOR]

While reversible histone modifications are linked to an ever-expanding range of biological functions, the demethylases for histone H4 lysine 20 and their potential regulatory roles remain unknown. Here we report that the PHD and Jumonji C (JmjC) domain-containing protein, PHF8, while using multiple substrates, including H3K9me1/2 and H3K27me2, also functions as an H4K20me1 demethylase. PHF8 is recruited to promoters by its PHD domain based on interaction with H3K4me2/3 and controls G1–S transition in conjunction with E2F1, HCF-1 (also known as HCFC1) and SET1A (also known as SETD1A), at least in part, by removing the repressive H4K20me1 mark from a subset of E2F1-regulated gene promoters. Phosphorylation-dependent PHF8 dismissal from chromatin in prophase is apparently required for the accumulation of H4K20me1 during early mitosis, which might represent a component of the condensin II loading process. Accordingly, the HEAT repeat clusters in two non-structural maintenance of chromosomes (SMC) condensin II subunits, N-CAPD3 and N-CAPG2 (also known as NCAPD3 and NCAPG2, respectively), are capable of recognizing H4K20me1, and ChIP-Seq analysis demonstrates a significant overlap of condensin II and H4K20me1 sites in mitotic HeLa cells. Thus, the identification and characterization of an H4K20me1 demethylase, PHF8, has revealed an intimate link between this enzyme and two distinct events in cell cycle progression. [ABSTRACT FROM AUTHOR]

DNA polymerase η (Polη) is unique among eukaryotic polymerases in its proficient ability for error-free replication through ultraviolet-induced cyclobutane pyrimidine dimers, and inactivation of Polη (also known as POLH) in humans causes the variant form of xeroderma pigmentosum (XPV). We present the crystal structures of Saccharomyces cerevisiae Polη (also known as RAD30) in ternary complex with a cis-syn thymine-thymine (T-T) dimer and with undamaged DNA. The structures reveal that the ability of Polη to replicate efficiently through the ultraviolet-induced lesion derives from a simple and yet elegant mechanism, wherein the two Ts of the T-T dimer are accommodated in an active site cleft that is much more open than in other polymerases. We also show by structural, biochemical and genetic analysis that the two Ts are maintained in a stable configuration in the active site via interactions with Gln 55, Arg 73 and Met 74. Together, these features define the basis for Polη’s action on ultraviolet-damaged DNA that is crucial in suppressing the mutagenic and carcinogenic consequences of sun exposure, thereby reducing the incidence of skin cancers in humans. [ABSTRACT FROM AUTHOR]

DNA polymerase δ (Pol δ) is a high-fidelity polymerase that has a central role in replication from yeast to humans. We present the crystal structure of the catalytic subunit of yeast Pol δ in ternary complex with a template primer and an incoming nucleotide. The structure, determined at 2.0-Å resolution, catches the enzyme in the act of replication, revealing how the polymerase and exonuclease domains are juxtaposed relative to each other and how a correct nucleotide is selected and incorporated. The structure also reveals the 'sensing' interactions near the primer terminus, which signal a switch from the polymerizing to the editing mode. Taken together, the structure provides a chemical basis for the bulk of DNA synthesis in eukaryotic cells and a framework for understanding the effects of cancer-causing mutations in Pol δ. [ABSTRACT FROM AUTHOR]

The 1,N6-ethenodeoxyadenosine (εdA) lesion is promutagenic and has been implicated in carcinogenesis. We show here that human Polι, a Y-family DNA polymerase, can promote replication through this lesion by proficiently incorporating a nucleotide opposite it. The structural basis of this action is rotation of the εdA adduct to the syn conformation in the Polι active site and presentation of its 'Hoogsteen edge' for hydrogen-bonding with incoming dTTP or dCTP. We also show that Polζ carries out the subsequent extension reaction and that efficiency of extension from εdA·T is notably higher than from εdA·C. Together, our studies reveal for the first time how the exocyclic εdA adduct is accommodated in a DNA polymerase active site, and they show that the combined action of Polι and Polζ provides for efficient and error-free synthesis through this potentially carcinogenic DNA lesion. [ABSTRACT FROM AUTHOR]

Almost all DNA polymerases show a strong preference for incorporating the nucleotide that forms the correct Watson-Crick base pair with the template base. In addition, the catalytic efficiencies with which any given polymerase forms the four possible correct base pairs are roughly the same. Human DNA polymerase-? (hPol?), a member of the Y family of DNA polymerases, is an exception to these rules. hPol? incorporates the correct nucleotide opposite a template adenine with a several hundred to several thousand fold greater efficiency than it incorporates the correct nucleotide opposite a template thymine, whereas its efficiency for correct nucleotide incorporation opposite a template guanine or cytosine is intermediate between these two extremes. Here we present the crystal structure of hPol? bound to a template primer and an incoming nucleotide. The structure reveals a polymerase that is ‘specialized’ for Hoogsteen base-pairing, whereby the templating base is driven to the syn conformation. Hoogsteen base-pairing offers a basis for the varied efficiencies and fidelities of hPol? opposite different template bases, and it provides an elegant mechanism for promoting replication through minor-groove purine adducts that interfere with replication. [ABSTRACT FROM AUTHOR]

The ability or inability of a DNA polymerase to extend a mispair directly affects the establishment of genomic mutations. We report here kinetic analyses of the ability of Dpo4, a Y-family polymerase from Sulfolobus solfataricus, to extend from all mispairs opposite a template G or T. Dpo4 is equally inefficient at extending these mispairs, which include, surprisingly, a G•T mispair expected to conform closely to Watson-Crick geometry. To elucidate the basis of this, we solved the structure of Dpo4 bound to G•T-mispaired primer template in the presence of an incoming nucleotide. As a control, we also determined the structure of Dpo4 bound to a matched A-T base pair at the primer terminus. The structures offer a basis for the low efficiency of Dpo4 in extending a G•T mispair: a reverse wobble that deflects the primer 3′-OH away from the incoming nucleotide. [ABSTRACT FROM AUTHOR]

Restriction endonuclease BglII completely encircles its target DNA, making contacts to both the major and minor grooves. To allow the DNA to enter and leave the binding cleft, the enzyme dimer has to rearrange. To understand how this occurs, we have solved the structure of the free enzyme at 2.3 Å resolution, as a complement to our earlier work on the BglII?DNA complex. Unexpectedly, the enzyme opens by a dramatic `scissor-like' motion, accompanied by a complete rearrangement of the α-helices at the dimer interface. Moreover, within each monomer, a set of residues ? a ‘lever’ ? lowers or raises to alternately sequester or expose the active site residues. Such an extreme difference in free versus complexed structures has not been reported for other restriction endonucleases. This elegant mechanism for capturing DNA may extend to other enzymes that encircle DNA. [ABSTRACT FROM AUTHOR]

Restriction endonucleases are remarkably resilient to alterations in their DNA binding specificity. To understand the basis of this immutability, we have determined the crystal structure of endonuclease BglII bound to its recognition sequence (AGATCT), at 1.5 Å resolution. We compare the structure of BglII to endonuclease BamHI, which recognizes a closely related DNA site (GGATCC). We show that both enzymes share a similar α/β core, but in BglII, the core is augmented by a β-sandwich domain that encircles the DNA to provide extra specificity. Remarkably, the DNA is contorted differently in the two structures, leading to different protein?DNA contacts for even the common base pairs. Furthermore, the BglII active site contains a glutamine in place of the glutamate at the general base position in BamHI, and only a single metal is found coordinated to the putative nucleophilic water and the phosphate oxygens. This surprising diversity in structures shows that different strategies can be successful in achieving site-specific recognition and catalysis in restriction endonucleases. [ABSTRACT FROM AUTHOR]

Presents research which described the structure of the bromodomain of the histone-acetyltransferase (HAT) co-activator P/CAF. Role of histone acetylation in chromatin remodelling and gene activation; Details about bromodomains and their structure; Interaction of bromodomains with acetylated lysine; Recognition of acetyl-lysine by P/CAF; Function of the bromodomain in gene transcription regulation.

Type II restriction enzymes are characterized by their remarkable specificity and simplicity. They require only divalent metals (such as Mg2+ or Mn2+) as cofactors to catalyze the hydrolysis of DNA. However, most of the structural work on endonucleases has been performed in the absence of metals, leaving unanswered questions about their mechanisms of DNA cleavage. Here we report structures of the endonuclease BamHI?DNA complex, determined in the presence of Mn2+ and Ca2+, that describe the enzyme at different stages of catalysis. Overall, the results support a two-metal mechanism of DNA cleavage for BamHI which is distinct from that of EcoRV. [ABSTRACT FROM AUTHOR]

Describes the structure of a homeotic (Hox) protein on its DNA-binding site in combination with a cofactor protein. Study of the Ultrabithorax (Ubx) protein, produced and functional in the posterior thoracic and interior abdominal segments of Drosophila; Ubx mutations that lead to posterior-to-central thorax transformations; Gain of an affinity and specificity by Hox proteins when they bind together with the homeoprotein Extradenticle.

Presents research which reported the crystal structure of the interferon regulatory factor (IRF)-1 region bound to the natural positive regulatory domain I (PRD I) DNA element from the interferon-beta promoter. Importance of IRF transcription factors and their DNA-binding region; Details about structure; Suggested basis for occurrence of GAAA repeats within IRF response elements; Assembly of interferon-beta enhancesome.

Cytarabine (AraC) is an essential chemotherapeutic for acute myeloid leukemia (AML) and resistance to this drug is a major cause of treatment failure. AraC is a nucleoside analog that differs from 2′-deoxycytidine only by the presence of an additional hydroxyl group at the C2′ position of the 2′-deoxyribose. The active form of the drug AraC 5′-triphosphate (AraCTP) is utilized by human replicative DNA polymerases to insert AraC at the 3′ terminus of a growing DNA chain. This impedes further primer extension and is a primary basis for the drug action. The Y-family translesion synthesis (TLS) DNA polymerase η (Polη) counteracts this barrier to DNA replication by efficient extension from AraC-terminated primers. Here, we provide high-resolution structures of human Polη with AraC incorporated at the 3′-primer terminus. We show that Polη can accommodate AraC at different stages of the catalytic cycle, and that it can manipulate the conformation of the AraC sugar via specific hydrogen bonding and stacking interactions. Taken together, the structures provide a basis for the ability of Polη to extend DNA synthesis from AraC terminated primers. [ABSTRACT FROM AUTHOR]

N1-methyl-deoxyadenosine (1-MeA) is formed by methylation of deoxyadenosine at the N1 atom. 1-MeA presents a block to replicative DNA polymerases due to its inability to participate in Watson-Crick (W-C) base pairing. Here we determine how human DNA polymerase-ι (Polι) promotes error-free replication across 1-MeA. Steady state kinetic analyses indicate that Polι is ~100 fold more efficient in incorporating the correct nucleotide T versus the incorrect nucleotide C opposite 1-MeA. To understand the basis of this selectivity, we determined ternary structures of Polι bound to template 1-MeA and incoming dTTP or dCTP. In both structures, template 1-MeA rotates to the syn conformation but pairs differently with dTTP versus dCTP. Thus, whereas dTTP partakes in stable Hoogsteen base pairing with 1-MeA, dCTP fails to gain a 'foothold' and is largely disordered. Together, our kinetic and structural studies show how Polι maintains discrimination between correct and incorrect incoming nucleotide opposite 1-MeA in preserving genome integrity. [ABSTRACT FROM AUTHOR]

We have solved the crystal structure of human DNA polymerase-ι (hPolι) bound to a template primer and incoming nucleotide, and shown that hPolι uses Hoogsteen base-pairing for normal DNA synthesis. Jimin Wang questions the space group we used and claims that Hoogsteen base-pairing is not supported by our electron-density maps. We show here that the issue of space group does not affect our conclusions and that our electron-density maps are clear in indicating that Hoogsteen and not Watson–Crick base-pairing occurs in this situation. [ABSTRACT FROM AUTHOR]

Presents a correction to the article "Eya Protein Phosphatase Activity Regulates Six1-Dach-Eya Transcriptional Effects in Mammalian Organogenesis," that was previously published in the 2003 issue of the periodical "Nature."

Many reactions in cells proceed via the sequestration of two DNA molecules in a synaptic complex. SfiI is a member of a growing family of restriction enzymes that can bind and cleave two DNA sites simultaneously. We present here the structures of tetrameric SfiI in complex with cognate DNA. The structures reveal two different binding states of SfiI: one with both DNA-binding sites fully occupied and the other with fully and partially occupied sites. These two states provide details on how SfiI recognizes and cleaves its target DNA sites, and gives insight into sequential binding events. The SfiI recognition sequence (GGCCNNNN↓NGGCC) is a subset of the recognition sequence of BglI (GCCNNNN↓NGGC), and both enzymes cleave their target DNAs to leave 3-base 3′ overhangs. We show that even though SfiI is a tetramer and BglI is a dimer, and there is little sequence similarity between the two enzymes, their modes of DNA recognition are unusually similar. [ABSTRACT FROM AUTHOR]