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DNA repair is now understood to play a key role in a variety of disease states, most notably cancer. Tools for studying DNA have typically relied on traditional biochemical methods which are often laborious and indirect. Efforts to study the biology and therapeutic relevance of DNA repair pathways can be limited by such methods. Recently, specific fluorescent probes have been developed to aid in the study of DNA repair. Fluorescent probes offer the advantage of being able to directly assay for DNA repair activity in a simple, mix-and-measure format. This review will summarize the distinct classes of probe designs and their potential utility in varied research and preclinical settings.

The number of DNA polymerases identified in each organism has mushroomed in the past two decades. Most newly found DNA polymerases specialize in translesion synthesis and DNA repair instead of replication. Although intrinsic error rates are higher for translesion and repair polymerases than for replicative polymerases, the specialized polymerases increase genome stability and reduce tumorigenesis. Reflecting the numerous types of DNA lesions and variations of broken DNA ends, translesion and repair polymerases differ in structure, mechanism, and function. Here, we review the unique and general features of polymerases specialized in lesion bypass, as well as in gap-filling and end-joining synthesis.

Genomic instability in disease and its fidelity in health depend on the DNA damage response (DDR), regulated in part from the complex of meiotic recombination 11 homolog 1 (MRE11), ATP-binding cassette-ATPase (RAD50), and phosphopeptide-binding Nijmegen breakage syndrome protein 1 (NBS1). The MRE11-RAD50-NBS1 (MRN) complex forms a multifunctional DDR machine. Within its network assemblies, MRN is the core conductor for the initial and sustained responses to DNA double-strand breaks, stalled replication forks, dysfunctional telomeres, and viral DNA infection. MRN can interfere with cancer therapy and is an attractive target for precision medicine. Its conformations change the paradigm whereby kinases initiate damage sensing. Delineated results reveal kinase activation, posttranslational targeting, functional scaffolding, conformations storing binding energy and enabling access, interactions with hub proteins such as replication protein A (RPA), and distinct networks at DNA breaks and forks. MRN biochemistry provides prototypic insights into how it initiates, implements, and regulates multifunctional responses to genomic stress.

Human nineteen complex (NTC) acts as a multimeric E3 ubiquitin ligase in DNA repair and splicing. The transfer of ubiquitin is mediated by Prp19-a homotetrameric component of NTC whose elongated coiled coils serve as an assembly axis for two other proteins called SPF27 and CDC5L. We find that Prp19 is inactive on its own and have elucidated the structural basis of its autoinhibition by crystallography and mutational analysis. Formation of the NTC core by stepwise assembly of SPF27, CDC5L, and PLRG1 onto the Prp19 tetramer enables ubiquitin ligation. Protein-protein crosslinking of NTC, functional assays in vitro, and assessment of its role in DNA damage response provide mechanistic insight into the organization of the NTC core and the communication between PLRG1 and Prp19 that enables E3 activity. This reveals a unique mode of regulation for a complex E3 ligase and advances understanding of its dynamics in various cellular pathways., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The authors describe a colorimetric method for the sensitive and selective detection of Pb(II). It is based on the use exonuclease I (Exo I), a Pb(II)-binding aptamer bound to gold nanoparticles (AuNPs), and a DNA strand that complementary to the aptamer. In the absence of Pb(II), the dsDNA on the AuNPs prevents aggregation of the AuNPs in the presence of NaCl. In the presence of Pb(II), however, the aptamer binds Pb(II) and complementary strand is released and digested by Exo I. As a result, the solution of AuNPs undergoes a color change from red to purple if salt is added to the sample. The assay is selective for Pb(II) and has a limit of detection as low as 2.4 nM. It was successfully applied to the determination of Pb(II) in spiked tap water. Graphical abstract Schematic presentation of the aptamer based method for Pb 2+ detection via salt-induced aggregation of gold nanoparticles and colorimetric quantitation.

The Mre11-Rad50 protein complex is an initial responder to sites of DNA double strand breaks. Many studies have shown that ATP binding to Rad50 causes global changes to the Mre11-Rad50 structure, which are important for DNA repair functions. Here we used methyl-based NMR spectroscopy on a series of mutants to describe a dynamic allosteric pathway within Rad50. Mutations result in changes in the side chain methyl group chemical environment that are correlated with altered nanosecond timescale dynamics. We also observe striking relationships between the magnitude of chemical shift perturbations and Rad50 and Mre11 activities. Together, these data suggest an equilibrium between a ground state and an "active" dimerization competent state of Rad50 that has locally altered structure and dynamics and is poised for ATP-induced dimerization and eventual ATP hydrolysis. Thus, this sparsely populated intermediate is critical for Mre11-Rad50-directed DNA double strand break repair.

A strategy is described for the detection of protein by using a cationic fluorescent conjugated polymer coupled with exonuclease I (Exo I). Taking streptavidin (SA) as model protein, it is observed that Exo I can digest single-stranded DNA conjugated with biotin and carboxyfluorescein (P1) if SA is absent. This leads to the formation of small nucleotide fragments and to weak fluorescence resonance energy transfer (FRET) from the polymer to P1. If, however, SA is present, the high affinity of SA and biotin prevents the digestion of P1 by Exo I. This results in the sorption of P1 on the surface of the polymer through strong electrostatic interaction. Hence, efficient FRET occurs from the fluorescent polymer to the fluorescent label of P1. Fluorescence is measured at an excitation wavelength of 370 nm, and emission is measured at two wavelengths (530 and 425 nm). The ratio of the two intensities (I 530 /I 425 ) is directly related to the concentration of SA. Under the optimal conditions, the assay has a detection limit of 1.3 ng·mL -1 . The method was also applied to image the folate receptor in HeLa cells, thus demonstrating the versatility of this strategy. Graphical abstract A fluorometric strategy is described for protein detection and cell imaging based on a cationic conjugated polymer (PFP) coupled with exonuclease I (Exo I) trigged fluorescence resonance energy transfer (FRET).

Human Pre-mRNA Processing factor 19 (hPRPF19) is an important component in human spliceosome machinery. hPRPF19 contains a WD40 repeats domain at its C-terminus, which is also conserved in yeast. Here we determined the crystal structure of the C-terminal WD40 repeat domain of hPRPF19 by X-ray crystallography. Our structural analysis revealed some significantly different structure features between the human and yeast Prp19 WD40 repeat domain. However, there are also conserved clusters of residues at the bottom surface of the fourth and the fifth WD40 repeats, which provides the important implication for the conserved Prp19 proteins in both human and yeast., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The DNA is cells is continuously exposed to reactive oxygen species resulting in toxic and mutagenic DNA damage. Although the repair of oxidative DNA damage occurs primarily through the base excision repair (BER) pathway, the nucleotide excision repair (NER) pathway processes some of the same lesions. In addition, damage tolerance mechanisms, such as recombination and translesion synthesis, enable cells to tolerate oxidative DNA damage, especially when BER and NER capacities are exceeded. Thus, disruption of BER alone or disruption of BER and NER in Saccharomyces cerevisiae leads to increased mutations as well as large-scale genomic rearrangements. Previous studies demonstrated that a particular region of chromosome II is susceptible to chronic oxidative stress-induced chromosomal rearrangements, suggesting the existence of DNA damage and/or DNA repair hotspots. Here we investigated the relationship between oxidative damage and genomic instability utilizing chromatin immunoprecipitation combined with DNA microarray technology to profile DNA repair sites along yeast chromosomes under different oxidative stress conditions. We targeted the major yeast AP endonuclease Apn1 as a representative BER protein. Our results indicate that Apn1 target sequences are enriched for cytosine and guanine nucleotides. We predict that BER protects these sites in the genome because guanines and cytosines are thought to be especially susceptible to oxidative attack, thereby preventing large-scale genome destabilization from chronic accumulation of DNA damage. Information from our studies should provide insight into how regional deployment of oxidative DNA damage management systems along chromosomes protects against large-scale rearrangements. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

Xeroderma pigmentosum (XP) complementation group A (XPA) is an essential scaffolding protein in the multiprotein nucleotide excision repair (NER) machinery. The interaction of XPA with DNA is a core function of this protein; a number of mutations in the DNA-binding domain (DBD) are associated with XP disease. Although structures of the central globular domain of human XPA and data on binding of DNA substrates have been reported, the structural basis for XPA's DNA-binding activity remains unknown. X-ray crystal structures of the central globular domain of yeast XPA (Rad14) with lesion-containing DNA duplexes have provided valuable insights, but the DNA substrates used for this study do not correspond to the substrates of XPA as it functions within the NER machinery. To better understand the DNA-binding activity of human XPA in NER, we used NMR to investigate the interaction of its DBD with a range of DNA substrates. We found that XPA binds different single-stranded/double-stranded junction DNA substrates with a common surface. Comparisons of our NMR-based mapping of binding residues with the previously reported Rad14-DNA crystal structures revealed similarities and differences in substrate binding between XPA and Rad14. This includes direct evidence for DNA contacts to the residues extending C-terminally from the globular core, which are lacking in the Rad14 construct. Moreover, mutation of the XPA residue corresponding to Phe-262 in Rad14, previously reported as being critical for DNA binding, had only a moderate effect on the DNA-binding activity of XPA. The DNA-binding properties of several disease-associated mutations in the DBD were investigated. These results suggest that for XPA mutants exhibiting altered DNA-binding properties, a correlation exists between the extent of reduction in DNA-binding affinity and the severity of symptoms in XP patients., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Fragment-based lead discovery has emerged as a leading drug development strategy for novel therapeutic targets. Although fragment-based drug discovery benefits immensely from access to atomic-resolution information, structure-based virtual screening has rarely been used to drive fragment discovery and optimization. Here, molecular docking of 0.3 million fragments to a crystal structure of cancer target MTH1 was performed. Twenty-two predicted fragment ligands, for which analogs could be acquired commercially, were experimentally evaluated. Five fragments inhibited MTH1 with IC 50 values ranging from 6 to 79 μM. Structure-based optimization guided by predicted binding modes and analogs from commercial chemical libraries yielded nanomolar inhibitors. Subsequently solved crystal structures confirmed binding modes predicted by docking for three scaffolds. Structure-guided exploration of commercial chemical space using molecular docking gives access to fragment libraries that are several orders of magnitude larger than those screened experimentally and can enable efficient optimization of hits to potent leads.

The scaffold protein X-ray repair cross-complementing 1 (XRCC1) interacts with multiple enzymes involved in DNA base excision repair and single-strand break repair (SSBR) and is important for genetic integrity and normal neurological function. One of the most important interactions of XRCC1 is that with polynucleotide kinase/phosphatase (PNKP), a dual-function DNA kinase/phosphatase that processes damaged DNA termini and that, if mutated, results in ataxia with oculomotor apraxia 4 (AOA4) and microcephaly with early-onset seizures and developmental delay (MCSZ). XRCC1 and PNKP interact via a high-affinity phosphorylation-dependent interaction site in XRCC1 and a forkhead-associated domain in PNKP. Here, we identified using biochemical and biophysical approaches a second PNKP interaction site in XRCC1 that binds PNKP with lower affinity and independently of XRCC1 phosphorylation. However, this interaction nevertheless stimulated PNKP activity and promoted SSBR and cell survival. The low-affinity interaction site required the highly conserved Rev1-interacting region (RIR) motif in XRCC1 and included three critical and evolutionarily invariant phenylalanine residues. We propose a bipartite interaction model in which the previously identified high-affinity interaction acts as a molecular tether, holding XRCC1 and PNKP together and thereby promoting the low-affinity interaction identified here, which then stimulates PNKP directly., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

RPA-coated single-stranded DNA (RPA-ssDNA), a nucleoprotein structure induced by DNA damage, promotes ATR activation and homologous recombination (HR). RPA is hyper-phosphorylated and ubiquitylated after DNA damage. The ubiquitylation of RPA by PRP19 and RFWD3 facilitates ATR activation and HR, but how it is stimulated by DNA damage is still unclear. Here, we show that RFWD3 binds RPA constitutively, whereas PRP19 recognizes RPA after DNA damage. The recruitment of PRP19 by RPA depends on PIKK-mediated RPA phosphorylation and a positively charged pocket in PRP19. An RPA32 mutant lacking phosphorylation sites fails to recruit PRP19 and support RPA ubiquitylation. PRP19 mutants unable to bind RPA or lacking ubiquitin ligase activity also fail to support RPA ubiquitylation and HR. These results suggest that RPA phosphorylation enhances the recruitment of PRP19 to RPA-ssDNA and stimulates RPA ubiquitylation through a process requiring both PRP19 and RFWD3, thereby triggering a phosphorylation-ubiquitylation circuitry that promotes ATR activation and HR., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

N-methyl-2,2-di(chloroethyl)amine (HN2) is a kind of bifunctional alkyltating agent, which can react with nucleophilic groups in DNA and/or protein to form HN2-bridged crosslinking of target molecules, such as DNA-protein crosslinkings (DPC). O 6 -methylguanine-DNA methyltransferase (MGMT) is a DNA damage repair enzyme which solely repairs alkyl adduct on DNA directly. However, MGMT was detected to act as a protein cross-linked with DNA via alkylation in presence of HN2, and unexpectedly turned into a DNA damage enhancer in the form of MGMT-DNA cross-link (mDPC). Present study aimed to explore the possible ways to lessen the incorporation of MGMT into DPC as well as to save it for DNA repair. To find out the influencing factors of mDPC formation and cleavage, human bronchial epithelial cell line 16HBE was exposed to HN2 and the factors related with MGMT expression and degradation were investigated. When c-Myc, a negative transcriptional factor of MGMT was inhibited by 10058-F4, MGMT expression and mDPC formation were increased, and more γ-H2AX was also detected. Sustained treatment with O 6 BG, a specific exogenous substrate and depleter of MGMT, could reduce the level of MGMT and mDPC formation. In contrast, a transient 1h pre-treatment of O 6 GB before HN2 exposure would cause a high MGMT and mDPC level. MGMT was increasingly ubiquitinated after HN2 exposure in a time-dependent manner. At the same time, MGMT was also SUMOylated with a downward time-dependent manner compared to its ubiquitination. Inhibitors of E1, E2 or E3 ligases of ubiqutination all led to the accumulation of mDPC and total-DPC (tDPC) with the difference as that mDPC was sensitive to E1 inhibitor while tDPC more sensitive to E2 and E3 inhibitor. Our results demonstrated the control of mDPC level could be realized through transcription inhibitory effect of c-Myc, O 6 GB application, and the acceleration of mDPC ubiquitination and subsequent degradation., (Copyright © 2017. Published by Elsevier B.V.)

Human exonuclease 1 (hEXO1) is a member of the 5'-nuclease superfamily and plays important roles in DNA repair. Along with acting as a 5'-exonuclease on blunt, gapped, nicked, and 3'-overhang DNAs, hEXO1 can also act as an endonuclease removing protruding 5'-single-stranded flaps from duplex ends. How hEXO1 and related 5'-nuclease human flap endonuclease 1 (hFEN1) are specific for discontinuous DNA substrates like 5'-flaps has been controversial. Here we report the first functional data that imply that hEXO1 threads the 5'-flap through a hole in the protein known as the helical arch, thereby excluding reactions of continuous single strands. Conjugation of bulky 5'-streptavidin that would "block" threading through the arch drastically slowed the hEXO1 reaction. In contrast, addition of streptavidin to a preformed hEXO1 5'-biotin flap DNA complex trapped a portion of the substrate in a highly reactive threaded conformation. However, another fraction behaves as if it were "blocked" and decayed very slowly, implying there were both threaded and unthreaded forms of the substrate present. The reaction of an unmodified hEXO1-flap DNA complex did not exhibit marked biphasic kinetics, suggesting a fast re-equilibration occurs that produces more threaded substrate when some decays. The finding that a threading mechanism like that used by hFEN1 is also used by hEXO1 unifies the mode of operation for members of the 5'-nuclease superfamily that act on discontinuous substrates. As with hFEN1, intrinsic disorder of the arch region of the protein may explain how flaps can be threaded without a need for a coupled energy source.

Human exonuclease 1 (hExo1) is a member of the RAD2/XPG structure-specific 5'-nuclease superfamily. Its dominant, processive 5'-3' exonuclease and secondary 5'-flap endonuclease activities participate in various DNA repair, recombination, and replication processes. A single active site processes both recessed ends and 5'-flap substrates. By initiating enzyme reactions in crystals, we have trapped hExo1 reaction intermediates that reveal structures of these substrates before and after their exo- and endonucleolytic cleavage, as well as structures of uncleaved, unthreaded, and partially threaded 5' flaps. Their distinctive 5' ends are accommodated by a small, mobile arch in the active site that binds recessed ends at its base and threads 5' flaps through a narrow aperture within its interior. A sequence of successive, interlocking conformational changes guides the two substrate types into a shared reaction mechanism that catalyzes their cleavage by an elaborated variant of the two-metal, in-line hydrolysis mechanism. Coupling of substrate-dependent arch motions to transition-state stabilization suppresses inappropriate or premature cleavage, enhancing processing fidelity. The striking reduction in flap conformational entropy is catalyzed, in part, by arch motions and transient binding interactions between the flap and unprocessed DNA strand. At the end of the observed reaction sequence, hExo1 resets without relinquishing DNA binding, suggesting a structural basis for its processivity., Competing Interests: The authors declare no conflict of interest.

Non-homologous end joining (NHEJ) repairs DNA double strand breaks in non-cycling eukaryotic cells. NHEJ relies on polynucleotide kinase/phosphatase (PNKP), which generates 5΄-phosphate/3΄-hydroxyl DNA termini that are critical for ligation by the NHEJ DNA ligase, LigIV. PNKP and LigIV require the NHEJ scaffolding protein, XRCC4. The PNKP FHA domain binds to the CK2-phosphorylated XRCC4 C-terminal tail, while LigIV uses its tandem BRCT repeats to bind the XRCC4 coiled-coil. Yet, the assembled PNKP-XRCC4-LigIV complex remains uncharacterized. Here, we report purification and characterization of a recombinant PNKP-XRCC4-LigIV complex. We show that the stable binding of PNKP in this complex requires XRCC4 phosphorylation and that only one PNKP protomer binds per XRCC4 dimer. Small angle X-ray scattering (SAXS) reveals a flexible multi-state complex that suggests that both the PNKP FHA and catalytic domains contact the XRCC4 coiled-coil and LigIV BRCT repeats. Hydrogen-deuterium exchange indicates protection of a surface on the PNKP phosphatase domain that may contact XRCC4-LigIV. A mutation on this surface (E326K) causes the hereditary neuro-developmental disorder, MCSZ. This mutation impairs PNKP recruitment to damaged DNA in human cells and provides a possible disease mechanism. Together, this work unveils multipoint contacts between PNKP and XRCC4-LigIV that regulate PNKP recruitment and activity within NHEJ., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Double strand breaks (DSBs) and interstrand crosslinks (ICLs) are toxic DNA lesions that can be repaired through multiple pathways, some of which involve shared proteins. One of these proteins, DNA Polymerase θ (Pol θ), coordinates a mutagenic DSB repair pathway named microhomology-mediated end joining (MMEJ) and is also a critical component for bypass or repair of ICLs in several organisms. Pol θ contains both polymerase and helicase-like domains that are tethered by an unstructured central region. While the role of the polymerase domain in promoting MMEJ has been studied extensively both in vitro and in vivo, a function for the helicase-like domain, which possesses DNA-dependent ATPase activity, remains unclear. Here, we utilize genetic and biochemical analyses to examine the roles of the helicase-like and polymerase domains of Drosophila Pol θ. We demonstrate an absolute requirement for both polymerase and ATPase activities during ICL repair in vivo. However, similar to mammalian systems, polymerase activity, but not ATPase activity, is required for ionizing radiation-induced DSB repair. Using a site-specific break repair assay, we show that overall end-joining efficiency is not affected in ATPase-dead mutants, but there is a significant decrease in templated insertion events. In vitro, Pol θ can efficiently bypass a model unhooked nitrogen mustard crosslink and promote DNA synthesis following microhomology annealing, although ATPase activity is not required for these functions. Together, our data illustrate the functional importance of the helicase-like domain of Pol θ and suggest that its tethering to the polymerase domain is important for its multiple functions in DNA repair and damage tolerance.

The Mre11-Rad50-Nbs1(Xrs2) (MRN/X) complex is critical for the repair and signaling of DNA double strand breaks. The catalytic core of MRN/X comprised of the Mre11 nuclease and Rad50 adenosine triphosphatase (ATPase) active sites dimerizes through association between the Rad50 ATPase catalytic domains and undergoes extensive conformational changes upon ATP binding. This ATP-bound 'closed' state promotes binding to DNA, tethering DNA ends and ATM activation, but prevents nucleolytic processing of DNA ends, while ATP hydrolysis is essential for Mre11 endonuclease activity at blocked DNA ends. Here we investigate the regulation of ATP hydrolysis as well as the interdependence of the two functional active sites. We find that double-stranded DNA stimulates ATP hydrolysis by hMRN over ∼20-fold in an end-dependent manner. Using catalytic site mutants to create Rad50 dimers with only one functional ATPase site, we find that both ATPase sites are required for the stimulation by DNA. MRN-mediated endonucleolytic cleavage of DNA at sites of protein adducts requires ATP hydrolysis at both sites, as does the stimulation of ATM kinase activity. These observations suggest that symmetrical engagement of the Rad50 catalytic head domains with ATP bound at both sites is important for MRN functions in eukaryotic cells., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Multiple primary melanoma (MPM) is a rare condition, whose genetic basis has not yet been clarified. Only 8-12% of MPM are due to germline mutations of CDKN2A. However, other genes (POT1, BRCA1/2, MC1R, MGMT) have been demonstrated to be involved in predisposition to this pathology.To our knowledge, this is the first family study based on two siblings with the rare coexistence of MPM and oculocutaneous albinism (OCA), an autosomal recessive disease characterized by the absence or decrease in pigmentation in the skin, hair, and eyes.In this study, we evaluated genes involved in melanoma predisposition (CDKN2A, CDK4, MC1R, MITF, POT1, RB1, MGMT, BRCA1, BRCA2), pathogenesis (BRAF, NRAS, PIK3CA, KIT, PTEN), skin/hair pigmentation (MC1R, MITF) and in immune pathways (CTLA4) to individuate alterations able to explain the rare onset of MPM and OCA in indexes and the transmission in their pedigree.From the analysis of the pedigree, we were able to identify a "protective" haplotype with respect to MPM, including MGMT p.I174V alteration. The second generation offspring is under strict follow up as some of them have a higher risk of developing MPM according to our model.

T4 polynucleotide kinase phosphatase (T4 PNKP) is a bifunctional tool enzyme with 5'-kinase and 3'-phosphatase activities. Considering its important roles in the repair of strand breaks, assay of T4 PNKP activity is of great importance. In this work, we proposed a novel label-free sensing strategy for T4 PNKP activity based on G-quadruplexe-thioflavin T fluorescent indicator. In the assay, we used a single stranded oligonucleotide with 3'-phosphoryl and 5'-phosphoryl ends as enzyme substrate. Upon the addition of T4 PNKP, the 3'-PO 3 of the substrate was changed to 3'-OH which initiated the polymerization in the presence of terminal deoxynucleotidyl transferase and G-rich dNTP substrates. The resultant elongated DNA can form G-quadruplex in the inducement of K + , resulting in strong fluorescence signal when using thioflavin T as a G-quadruplex-specific light-up fluorescent probe. The detection limit of this method is as low as 0.2U/mL. Additionally, the inhibition of T4 PNKP activity by the inhibitor heparin is demonstrated. This method is easy and convenient to operate in homogeneous solution, and the whole assay process can be completed in a single tube., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Coordination between DNA replication and DNA repair ensures maintenance of genome integrity, which is lost in cancer cells. Emerging evidence has linked homologous recombination (HR) proteins RAD51, BRCA1 and BRCA2 to the stability of nascent DNA. This function appears to be distinct from double-strand break (DSB) repair and is in part due to the prevention of MRE11-mediated degradation of nascent DNA at stalled forks. The role of RAD51 in fork protection resembles the activity described for its prokaryotic orthologue RecA, which prevents nuclease-mediated degradation of DNA and promotes replication fork restart in cells challenged by DNA-damaging agents. Here, we examine the mechanistic aspects of HR-mediated fork protection, addressing the crosstalk between HR and replication proteins., (© 2017 Federation of European Biochemical Societies.)

The Rad50 hook interface is crucial for assembly and various functions of the Mre11 complex. Previous analyses suggested that Rad50 molecules interact within (intracomplex) or between (intercomplex) dimeric complexes. In this study, we determined the structure of the human Rad50 hook and coiled-coil domains. The data suggest that the predominant structure is the intracomplex, in which the two parallel coiled coils proximal to the hook form a rod shape, and that a novel interface within the coiled-coil domains of Rad50 stabilizes the interaction of Rad50 protomers in the dimeric assembly. In yeast, removal of the coiled-coil interface compromised Tel1 activation without affecting DNA repair, while simultaneous disruption of that interface and the hook phenocopied a null mutation. The results demonstrate that the hook and coiled-coil interfaces coordinately promote intracomplex assembly and define the intracomplex as the functional form of the Mre11 complex.

Shelterin is a six-protein complex (TRF1, TRF2, POT1, RAP1, TIN2, and TPP1) that also functions in smaller subsets in regulation and protection of human telomeres. Two closely related proteins, TRF1 and TRF2, make high-affinity contact directly with double-stranded telomeric DNA and serve as a molecular platform. Protein TIN2 binds to TRF1 and TRF2 dimer-forming domains, whereas Apollo makes interaction only with TRF2. To elucidate the molecular basis of these interactions, we employed molecular dynamics (MD) simulations of TRF1 TRFH -TIN2 TBM and TRF2 TRFH -TIN2 TBM /Apollo TBM complexes and of the isolated proteins. MD enabled a structural and dynamical comparison of protein-peptide complexes including H-bond interactions and interfacial residues that may regulate TRF protein binding to the given peptides, especially focusing on interactions described in crystallographic data. Residues with a selective function in both TRF1 TRFH and TRF2 TRFH and forming a stable hydrogen bond network with TIN2 TBM or Apollo TBM peptides were traced. Our study revealed that TIN2 TBM forms a well-defined binding mode with TRF1 TRFH as compared to TRF2 TRFH , and that the binding pocket of TIN2 TBM is deeper for TRF2 TRFH protein than Apollo TBM . The MD data provide a basis for the reinterpretation of mutational data obtained in crystallographic work for the TRF proteins. Together, the previously determined X-ray structure and our MD provide a detailed view of the TRF-peptide binding mode and the structure of TRF1/2 binding pockets. Particular TRF-peptide interactions are very specific for the formation of each protein-peptide complex, identifying TRF proteins as potential targets for the design of inhibitors/drugs modulating telomere machinery for anticancer therapy.

Non-homologous end-joining (NHEJ) is the dominant means of repairing chromosomal DNA double strand breaks (DSBs), and is essential in human cells. Fifteen or more proteins can be involved in the detection, signalling, synapsis, end-processing and ligation events required to repair a DSB, and must be assembled in the confined space around the DNA ends. We review here a number of interaction points between the core NHEJ components (Ku70, Ku80, DNA-PKcs, XRCC4 and Ligase IV) and accessory factors such as kinases, phosphatases, polymerases and structural proteins. Conserved protein-protein interaction sites such as Ku-binding motifs (KBMs), XLF-like motifs (XLMs), FHA and BRCT domains illustrate that different proteins compete for the same binding sites on the core machinery, and must be spatially and temporally regulated. We discuss how post-translational modifications such as phosphorylation, ADP-ribosylation and ubiquitinylation may regulate sequential steps in the NHEJ pathway or control repair at different types of DNA breaks., (© 2017 WILEY Periodicals, Inc.)

The human MutT homolog 1 (hMTH1, human NUDT1) hydrolyzes oxidatively damaged nucleoside triphosphates and is the main enzyme responsible for nucleotide sanitization. hMTH1 recently has received attention as an anticancer target because hMTH1 blockade leads to accumulation of oxidized nucleotides in the cell, resulting in mutations and death of cancer cells. Unlike Escherichia coli MutT, which shows high substrate specificity for 8-oxoguanine nucleotides, hMTH1 has broad substrate specificity for oxidized nucleotides, including 8-oxo-dGTP and 2-oxo-dATP. However, the reason for this broad substrate specificity remains unclear. Here, we determined crystal structures of hMTH1 in complex with 8-oxo-dGTP or 2-oxo-dATP at neutral pH. These structures based on high quality data showed that the base moieties of two substrates are located on the similar but not the same position in the substrate binding pocket and adopt a different hydrogen-bonding pattern, and both triphosphate moieties bind to the hMTH1 Nudix motif ( i.e. the hydrolase motif) similarly and align for the hydrolysis reaction. We also performed kinetic assays on the substrate-binding Asp-120 mutants (D120N and D120A), and determined their crystal structures in complex with the substrates. Analyses of bond lengths with high-resolution X-ray data and the relationship between the structure and enzymatic activity revealed that hMTH1 recognizes the different oxidized nucleotides via an exchange of the protonation state at two neighboring aspartate residues (Asp-119 and Asp-120) in its substrate binding pocket. To our knowledge, this mechanism of broad substrate recognition by enzymes has not been reported previously and may have relevance for anticancer drug development strategies targeting hMTH1., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

B-Myb, a highly conserved member of the Myb transcription factor family, is expressed ubiquitously in proliferating cells and controls the cell cycle dependent transcription of G2/M-phase genes. Deregulation of B-Myb has been implicated in oncogenesis and loss of genomic stability. We have identified B-Myb as a novel interaction partner of the Mre11-Rad50-Nbs1 (MRN) complex, a key player in the repair of DNA double strand breaks. We show that B-Myb directly interacts with the Nbs1 subunit of the MRN complex and is recruited transiently to DNA-damage sites. In response to DNA-damage B-Myb is phosphorylated by protein kinase GSK3β and released from the MRN complex. A B-Myb mutant that cannot be phosphorylated by GSK3β disturbs the regulation of pro-mitotic B-Myb target genes and leads to inappropriate mitotic entry in response to DNA-damage. Overall, our work suggests a novel function of B-Myb in the cellular DNA-damage signalling., Competing Interests: The authors declare no competing financial interests.

Mammalian cells express two homologs of yeast Rad23, the so-called homolog of Rad23 (HR23) proteins. The HR23 proteins were identified more than two decades ago as factors involved in initiation of global genome nucleotide excision repair (GG-NER) along with their interacting partner, xeroderma pigmentosum group C (XPC) protein. Because the HR23 genes encode proteins harboring ubiquitin-like (UBL) domains at their N-termini and two ubiquitin-associated (UBA) domains in their central- and C-terminal regions, the link between HR23 proteins and proteolytic degradation has been widely explored by several methods, including yeast two-hybrid screening and co-affinity purification. To date, various HR23 protein partners have been identified, and these proteins are involved not only in DNA repair, but also in ubiquitin-dependent protein degradation, transcriptional regulation, and cell cycle control. In addition, establishment of mouse strains lacking the HR23 genes and RNA silencing of these genes in human cells demonstrated their significance in animal development and cell growth. Through these studies, the functional differences between the two HR23 proteins have been gradually revealed. Furthermore, recent comprehensive proteomic analyses will help to elucidate the functional protein-protein networks involving the HR23 proteins., (Copyright © 2016 Elsevier B.V. All rights reserved.)

MTH1 can hydrolyze oxidized nucleotides and is required for cancer survival. The IC 50 values were 0.8 nM for TH287 with a methyl substitution, 5.0 nM for TH588 with a cyclopropyl substitution, and 2.1 μM for TH650 with an oxetanyl substitution. Thus, it is very significant to understand inhibitory mechanisms of these structurally similar compounds against MTH1 and influences of the substituent on the bioactivities. Our MD researches indicate that TH287 maintains significant hydrogen bonds with Asn33 and Asp119, stabilizes the binding site, and induces MTH1 adopt a closed motion, leading to a high inhibitory activity. When bound with TH588, the binding site can be partially stabilized and take a semi-closed state, which is because the cyclopropyl group in TH588 has larger steric hindrance than a methyl group in TH287. So TH588 has a slightly reduced inhibitory activity compared to TH287. TH650 induces greater conformation fluctuations than TH588 and the binding site adopts an opening state, which is caused by the large bulk of oxetanyl group and the interference of solvent on the oxetanyl substituent, leading to the lowest inhibitory activity. Thus, the inhibitory activity follows a TH287 > TH588 > TH650 trend, which well matches with the experimental finding.

DNA repair complexes play crucial roles in maintaining genome integrity, which is essential for the survival of an organism. The understanding of their modes of action is often obscure due to limited structural knowledge. Structural characterizations of these complexes are often challenging due to a poor protein production yield, a conformational flexibility, and a relatively high molecular mass. Single-particle electron microscopy (EM) has been successfully applied to study some of these complexes as it requires low amount of samples, is not limited by the high molecular mass of a protein or a complex, and can separate heterogeneous assemblies. Recently, near-atomic resolution structures have been obtained with EM owing to the advances in technology and image processing algorithms. In this chapter, we review the EM methodology of obtaining three-dimensional reconstructions of macromolecular complexes and provide a workflow that can be applied to DNA repair complex assemblies., (© 2017 Elsevier Inc. All rights reserved.)

The nonhomologous DNA end-joining (NHEJ) pathway is a key mechanism for repairing dsDNA breaks that occur often in eukaryotic cells. In the simplest model, these breaks are first recognized by Ku, which then interacts with other NHEJ proteins to improve their affinity at DNA ends. These include DNA-PK cs and Artemis for trimming the DNA ends; DNA polymerase μ and λ to add nucleotides; and the DNA ligase IV complex to ligate the ends with the additional factors, XRCC4 (X-ray repair cross-complementing protein 4), XLF (XRCC4-like factor/Cernunos), and PAXX (paralog of XRCC4 and XLF). In vivo studies have demonstrated the degrees of importance of these NHEJ proteins in the mechanism of repair of dsDNA breaks, but interpretations can be confounded by other cellular processes. In vitro studies with NHEJ proteins have been performed to evaluate the nucleolytic resection, polymerization, and ligation steps, but a complete system has been elusive. Here we have developed a NHEJ reconstitution system that includes the nuclease, polymerase, and ligase components to evaluate relative NHEJ efficiency and analyze ligated junctional sequences for various types of DNA ends, including blunt, 5' overhangs, and 3' overhangs. We find that different dsDNA end structures have differential dependence on these enzymatic components. The dependence of some end joining on only Ku and XRCC4·DNA ligase IV allows us to formulate a physical model that incorporates nuclease and polymerase components as needed., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

A new method for predicting the energy contributions to substrate binding and to specificity has been developed. Conventional global optimization methods do not permit the subtle effects responsible for these properties to be modeled with sufficient precision to allow confidence to be placed in the results, but by making simple alterations to the model, the precisions of the various energies involved can be improved from about ±2 kcal mol -1 to ±0.1 kcal mol -1 . This technique was applied to the oxidized nucleotide pyrophosphohydrolase enzyme MTH1. MTH1 is unusual in that the binding and reaction sites are well separated-an advantage from a computational chemistry perspective, as it allows the energetics involved in docking to be modeled without the need to consider any issues relating to reaction mechanisms. In this study, two types of energy terms were investigated: the noncovalent interactions between the binding site and the substrate, and those responsible for discriminating between the oxidized nucleotide 8-oxo-dGTP and the normal dGTP. Both of these were investigated using the semiempirical method PM7 in the program MOPAC. The contributions of the individual residues to both the binding energy and the specificity of MTH1 were calculated by simulating the effect of mutations. Where comparisons were possible, all calculated results were in agreement with experimental observations. This technique provides fresh insight into the binding mechanism that enzymes use for discriminating between possible substrates., Competing Interests: Compliance with ethical standards Disclaimer This work is solely the responsibility of the author and does not necessarily represent the official views of the National Institutes of Health.

Allostery is conformation regulation by propagating a signal from one site to another distal site. This study focuses on the long-range communication in DNA mismatch repair proteins MutS and its homologs where intramolecular signaling has to travel over 70 Å to couple lesion detection to ATPase activity and eventual downstream repair. Using dynamic network analysis based on extensive molecular dynamics simulations, multiple preserved communication pathways were identified that would allow such long-range signaling. The pathways appear to depend on the nucleotides bound to the ATPase domain as well as the type of DNA substrate consistent with previously proposed functional cycles of mismatch recognition and repair initiation by MutS and homologs. A mechanism is proposed where pathways are switched without major conformational rearrangements allowing for efficient long-range signaling and allostery., Competing Interests: The authors have declared that no competing interests exist.

Skin cancer is among one of the most common human malignancies wide-spread world-over with mortality statistics rising continuously at an alarming rate. The increasing frequency of these malignancies has marked the need for adopting effective treatment plan coupled with better and site-specific delivery options for the desired therapeutic agent's availability at the affected site. The concurrent delivery approaches to cancerous tissues are under constant challenge and, as a result, are evolving and gaining advancements in terms of delivery modes, therapeutic agents and site-specificity of the therapeutics delivery. The lipid-based liposomal drug delivery is an attractive and emerging option, and which is meticulously shaping up beyond a threshold level to a promising, and viable route for the effective delivery of therapeutic agents and other required injuctions to the skin cancer. An update on liposomal delivery of chemotherapeutic agents, natural-origin compounds, photosensitizer, and DNA repair enzymes as well as other desirable and typical delivery modes employed in drug delivery and in the treatment of skin cancers is discussed in details. Moreover, liposomal delivery of nucleic acid-based therapeutics, i.e., small interfering RNA (siRNA), mRNA therapy, and RGD-linked liposomes are among the other promising novel technology under constant development. The current clinical applicability, viable clinical plans, future prospects including transport feasibility of delivery vesicles and imaging techniques in conjunction with the therapeutic agents is also discussed. The ongoing innovations in liposomal drug delivery technology for skin cancers hold promise for further development of the methodology for better, more effective and site-specific delivery as part of the better treatment plan by ensuring faster drug transport, better and full payload delivery with enough and required concentration of the dose., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Mono-ADP-ribosylation is a dynamic posttranslational modification (PTM) with important roles in signaling. Mammalian proteins that recognize or hydrolyze mono-ADP-ribosylated proteins have been described. We report the synthesis of ADP-ribosylated peptides from the proteins histone H2B, RhoA and, HNP-1. An innovative procedure was applied that makes use of pre-phosphorylated amino acid building blocks. Binding assays revealed that the macrodomains of human MacroD2 and TARG1 exhibit distinct specificities for the different ADP-ribosylated peptides, thus showing that the sequence surrounding ADP-ribosylated residues affects the substrate selectivity of macrodomains., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We examined the interaction of polycyclic hydrocarbons (PAHs) like benzo-α-pyrene (BaP), chrysene, and their metabolites 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene,9,10-oxide (BPDE) and chrysene 1,2-diol-3,4-epoxide-2 (CDE), with the enzymes involved in DNA repair. We investigated interaction of 120 enzymes with PAHs and screened out 40 probable targets among DNA repair enzymes, on the basis of higher binding energy than positive control. Out of which, 20 enzymes lose their function in the presence of BaP, chrysene, and their metabolites, which may fetter DNA repair pathways resulting in damage accumulation and finally leading to cancer formation. We propose the use of nanoparticles as a guardian against the PAH's induced toxicity. PAHs enter the cell via aryl hydrocarbon receptor (AHR). TiO2 NP showed a much higher docking score with AHR (12,074) as compared with BaP and chrysene with AHR (4,600 and 4,186, respectively), indicating a preferential binding of TiO2 NP with the AHR. Further, docking of BaP and chrysene with the TiO2 NP bound AHR complex revealed their strong adsorption on TiO2 NP itself, and not on their original binding site (at AHR). TiO2 NPs thereby prevent the entry of PAHs into the cell via AHR and hence protect cells against the deleterious effects induced by PAHs., (© 2015 International Union of Biochemistry and Molecular Biology, Inc.)

Modern computational chemistry methods provide a powerful tool for use in refining the geometry of proteins determined by X-ray crystallography. Specifically, computational methods can be used to correctly place hydrogen atoms unresolved by this experimental method and improve bond geometry accuracy. Using the semiempirical method PM7, the structure of the nucleotide-sanitizing enzyme MTH1, complete with hydrolyzed substrate 8-oxo-dGMP, was optimized and the resulting geometry compared with the original X-ray structure of MTH1. After determining hydrogen atom placement and the identification of ionized sites, the charge distribution in the binding site was explored. Where comparison was possible, all the theoretical predictions were in good agreement with experimental observations. However, when these were combined with additional predictions for which experimental observations were not available, the result was a new and alternative description of the substrate-binding site interaction. An estimate was made of the strengths and weaknesses of the PM7 method for modeling proteins on varying scales, ranging from overall structure to individual interatomic distances. An attempt to correct a known fault in PM7, the under-estimation of steric repulsion, is also described. This work sheds light on the specificity of the enzyme MTH1 toward the substrate 8-oxo-dGTP; information that would facilitate drug development involving MTH1. Graphical Abstract Overlay of the backbone traces of the two MTH1 protein chains (green and orange respectively) in PDB 3ZR0 and the equivalent PM7 structures (magenta and cyan respectively) each optimized separately.

Genome integrity is vital to cellular homeostasis and its forfeiture is linked to deleterious consequences-cancer, immunodeficiency, genetic disorders and premature aging. The human ubiquitin ligase Pso4/Prp19 has emerged as a critical component of multiple DNA damage response (DDR) signaling networks. It not only senses DNA damage, binds double-stranded DNA in a sequence-independent manner, facilitates processing of damaged DNA, promotes DNA end joining, regulates replication protein A (RPA2) phosphorylation and ubiquitination at damaged DNA, but also regulates RNA splicing and mitotic spindle formation in its integral capacity as a scaffold for a multimeric core complex. Accordingly, by virtue of its regulatory and structural interactions with key proteins critical for genome integrity-DNA double-strand break (DSB) repair, DNA interstrand crosslink repair, repair of stalled replication forks and DNA end joining-it fills a unique niche in restoring genomic integrity after multiple types of DNA damage and thus has a vital role in maintaining chromatin integrity and cellular functions. These properties may underlie its ability to thwart replicative senescence and, not surprisingly, have been linked to the self-renewal and colony-forming ability of murine hematopoietic stem cells. This review highlights recent advances in hPso4 research that provides a fascinating glimpse into the pleiotropic activities of a ubiquitously expressed multifunctional E3 ubiquitin ligase.

The αββα metallo β-lactamase (MBL) fold (MBLf) was first observed in bacterial enzymes that catalyze the hydrolysis of almost all β-lactam antibiotics, but is now known to be widely distributed. The MBL core protein fold is present in human enzymes with diverse biological roles, including cell detoxification pathways and enabling resistance to clinically important anticancer medicines. Human (h)MBLf enzymes can bind metals, including zinc and iron ions, and catalyze a range of chemically interesting reactions, including both redox (e.g., ETHE1) and hydrolytic processes (e.g., Glyoxalase II, SNM1 nucleases, and CPSF73). With a view to promoting basic research on MBLf enzymes and their medicinal targeting, here we summarize current knowledge of the mechanisms and roles of these important molecules., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Cockayne syndrome is caused by biallelic ERCC8 (CSA) or ERCC6 (CSB) mutations and is characterized by growth restriction, microcephaly, developmental delay, and premature pathological aging. Typically affected patients also have dermal photosensitivity. Although Cockayne syndrome is considered a DNA repair disorder, patients with UV-sensitive syndrome, with ERCC8 (CSA) or ERCC6 (CSB) mutations have indistinguishable DNA repair defects, but none of the extradermal features of Cockayne syndrome. We report novel missense mutations affecting a conserved loop in the ERCC6 (CSB) protein, associated with the Cockayne syndrome phenotype. Indeed, the amino acid sequence of this loop is more highly conserved than the adjacent helicase motifs V and VI, suggesting that this is a crucial structural component of the SWI/SNF family of proteins, to which ERCC6 (CSB) belongs. These comprise two RecA-like domains, separated by an interdomain linker, which interact through helicase motif VI. As the observed mutations are likely to act through destabilizing the tertiary protein structure, this prompted us to re-evaluate ERCC6 (CSB) mutation data in relation to the structure of SWI/SNF proteins. Our analysis suggests that antimorphic mutations cause Cockayne syndrome and that biallelic interdomain linker deletions produce more severe phenotypes. Based on our observations, we propose that further investigation of the pathogenic mechanisms underlying Cockayne syndrome should focus on the effect of antimorphic rather than null ERCC6 (CSB) mutations., (© 2016 Wiley Periodicals, Inc.)

Polyubiquitination, a critical protein post-translational modification, signals for a diverse set of cellular events via the different isopeptide linkages formed between the C terminus of one ubiquitin (Ub) and the ɛ-amine of K6, K11, K27, K29, K33, K48, or K63 of a second Ub. We assembled di-ubiquitins (Ub2) comprising every lysine linkage and examined them biochemically and structurally. Of these, K27-Ub2 is unique as it is not cleaved by most deubiquitinases. As this remains the only structurally uncharacterized lysine linkage, we comprehensively examined the structures and dynamics of K27-Ub2 using nuclear magnetic resonance, small-angle neutron scattering, and in silico ensemble modeling. Our structural data provide insights into the functional properties of K27-Ub2, in particular that K27-Ub2 may be specifically recognized by K48-selective receptor UBA2 domain from proteasomal shuttle protein hHR23a. Binding studies and mutagenesis confirmed this prediction, further highlighting structural/recognition versatility of polyubiquitins and the potential power of determining function from elucidation of conformational ensembles., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Exonuclease 1 (EXO1) is required for both DNA repair and meiosis. Inactivation of EXO1 gene in mice leads to infertility. This study aimed to investigate whether variants in the EXO1 gene contribute to human premature ovarian failure (POF). The coding region of EXO1 was sequenced in 186 Han Chinese patients with non-syndromic POF. No plausible mutation was detected. The results suggest that mutations in the coding region of EXO1 may not be responsible for POF in Han Chinese women., (Copyright © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

As one of the most successful anticancer drugs, crizotinib is found to be efficient in the suppression of MTH1, a new therapeutic target for RAS-dependent cancers. Deep analysis shows that stereospecificity is prevalent in the binding of crizotinib to MTH1, where the target is more preferred to bind with the (S)-enantiomer of crizotinib. Surprisingly, very similar binding modes were found for the two enantiomers (Huber et al. Nature 2014, 508, 222-227), which puzzled us to ask a question as to why such a subtle structural variation could lead to so large of a binding affinity difference. Thereafter, by using advanced all-atom molecular dynamics simulations, we characterized the free energy surfaces of the binding/unbinding processes of the (S) and (R)-crizotinib enantiomers to/from MTH1. Interestingly, we found that rather than the induced-fit process, which is prevalent in drug selectivity and specificity (Wilson et al. Science 2015, 347, 882-886), the directly binding process has dominated impact on the binding affinity difference of the enantiomers, implying a common mechanism of stereoselectivity of enantiomers.

The machinery of DNA mismatch repair enzymes is highly conserved in evolution. The process is initiated by recognition of a DNA mismatch, and validated by ATP and the presence of a processivity clamp or a methylation mark. Several events in MMR promote conformational changes that lead to progression of the repair process. Here we discuss functional conformational changes in the MMR proteins and we compare the enzymes to paralogs in other systems., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

Cockayne syndrome (CS) is a recessive disorder that results in deficiencies in transcription-coupled nucleotide excision repair (TC-NER), a subpathway of nucleotide excision repair, and cells from CS patients exhibit hypersensitivity to UV light. CS group B protein (CSB), which is the gene product of one of the genes responsible for CS, belongs to the SWI2/SNF2 DNA-dependent ATPase family and has an ATPase domain and an ubiquitin-binding domain (UBD) in the central region and the C-terminal region, respectively. The C-terminal region containing the UBD is essential for the functions of CSB. In this study, we generated several CSB deletion mutants and analyzed the functions of the C-terminal region of CSB in TC-NER. Not only the UBD but also the C-terminal 30-amino acid residues were required for UV light resistance and TC-NER. This region was needed for the interaction of CSB with RNA polymerase II, the translocation of CS group A protein to the nuclear matrix, and the association of CSB with chromatin after UV irradiation. CSB was modified by small ubiquitin-like modifier 2/3 in a UV light-dependent manner. This modification was abolished in a CSB mutant lacking the C-terminal 30 amino acid residues. However, the substitution of lysine residues in this region with arginine did not affect SUMOylation or TC-NER. By contrast, substitution of a lysine residue in the N-terminal region with arginine decreased SUMOylation and resulted in cells with defects in TC-NER. These results indicate that both the most C-terminal region and SUMOylation are important for the functions of CSB in TC-NER., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The human SNM1A and SNM1B/Apollo proteins are members of an extended family of eukaryotic nuclease containing a motif related to the prokaryotic metallo-β-lactamase (MBL) fold. SNM1A is a key exonuclease during replication-dependent and transcription-coupled interstrand crosslink repair, while SNM1B/Apollo is required for maintaining telomeric overhangs. Here, we report the crystal structures of SNM1A and SNM1B at 2.16 Å. While both proteins contain a typical MBL-β-CASP domain, a region of positive charge surrounds the active site of SNM1A, which is absent in SNM1B and explains the greater apparent processivity of SNM1A. The structures of both proteins also reveal a putative, wide DNA-binding groove. Extensive mutagenesis of this groove, coupled with detailed biochemical analysis, identified residues that did not impact on SNM1A catalytic activity, but drastically reduced its processivity. Moreover, we identified a key role for this groove for efficient digestion past DNA interstrand crosslinks, facilitating the key DNA repair reaction catalysed by SNM1A. Together, the architecture and dimensions of this groove, coupled to the surrounding region of high positive charge, explain the remarkable ability of SNM1A to accommodate and efficiently digest highly distorted DNA substrates, such as those containing DNA lesions., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Variations in mismatch repair (MMR) system genes are causative of Lynch syndrome and other cancers. Thousands of variants have been identified in MMR genes, but the clinical relevance is known for only a small proportion. Recently, the InSiGHT group classified 2,360 MMR variants into five classes. One-third of variants, majority of which is nonsynonymous variants, remain to be of uncertain clinical relevance. Computational tools can be used to prioritize variants for disease relevance investigations. Previously, we classified 248 MMR variants as likely pathogenic and likely benign using PON-MMR. We have developed a novel tool, PON-MMR2, which is trained on a larger and more reliable dataset. In performance comparison, PON-MMR2 outperforms both generic tolerance prediction methods as well as methods optimized for MMR variants. It achieves accuracy and MCC of 0.89 and 0.78, respectively, in cross-validation and 0.86 and 0.69, respectively, on an independent test dataset. We classified 354 class 3 variants in InSiGHT database as well as all possible amino acid substitutions in four MMR proteins. Likely harmful variants mainly appear in the protein core, whereas likely benign variants are on the surface. PON-MMR2 is a highly reliable tool to prioritize variants for functional analysis. It is freely available at http://structure.bmc.lu.se/PON-MMR2/., (© 2015 WILEY PERIODICALS, INC.)

Human AlkB homolog 2 (ALKBH2) is a DNA repair enzyme that catalyzes the direct reversal of DNA methylation damage through oxidative demethylation. While ALKBH2 colocalizes with proliferating cell nuclear antigen (PCNA) in DNA replication foci, it remains unknown whether these two proteins alone form a complex or require additional components for interaction. Here, we demonstrate that ALKBH2 can directly interact with PCNA independent from other cellular factors, and we identify the hydrophobic pocket of PCNA as the key domain mediating this interaction. Moreover, we find that PCNA association with ALKBH2 increases significantly during DNA replication, suggesting that ALKBH2 forms a cell-cycle dependent complex with PCNA. Intriguingly, we show that an ALKBH2 germline variant, as well as a variant found in cancer, display altered interaction with PCNA. Our studies reveal the ALKBH2 binding interface of PCNA and indicate that both germline and somatic ALKBH2 variants could have cellular effects on ALKBH2 function in DNA repair., Competing Interests: The authors declare that there are no conflicts of interest., (Copyright © 2015 Elsevier B.V. All rights reserved.)