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1.
    
DNA interstrand cross-links (ICL)-inducing agents such as cisplatin, mitomycin C (MMC) and nitrogen mustards are widely used as potent antitumor drugs. Although ICL repair mechanism is not yet well characterized in mammalian cells, this pathway is thought to involve a sequential action of nucleotide excision repair (NER) and homologous recombination (HR). The importance of unraveling ICL repair pathways is highlighted by the hypersensitivity to ICL-inducing agents in cells of patients with the genetic disease Fanconi anemia (FA) and in cells mutated in the Breast Cancer susceptibility genes BRCA1 and BRCA2. To better characterize the involvement of HR in the sensitivity to ICL-inducing agents, we examined spontaneous and ICL-induced HR in rodent FA-like V-H4 cells. In this report, we show that MMC-hypersensitive V-H4 cells exhibit an increased spontaneous homology-directed repair (HDR) activity compared to the resistant V79 parental cells. Elevated HDR activity results mainly in increased conservative Rad51-dependent recombination, without affecting non-conservative single-strand annealing process (SSA). We also show that HDR activity is enhanced following MMC treatment in parental cells, but not in rodent FA-like V-H4 cells. Moreover, our data indicate that Rad51 foci formation is significantly delayed in these FA-like cells in response to crosslinking agent. These findings provide evidence for an impairment of HR control in V-H4 cells and emphasize the involvement of the FA pathway in HR-mediated repair.  相似文献   

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The base excision repair (BER) pathway is mainly responsible for the repair of a vast number of non-bulky lesions produced by alkylation, oxidation or deamination of bases. DNA glycosylases are the key enzymes that recognize damaged bases and initiate BER by catalyzing the cleavage of the N-glycosylic bond between the base and the sugar. Many of the mammalian DNA glycosylases have been identified by a combination of biochemical and bioinformatics analysis. Thus, a mammalian family of three proteins (NEIL1, NEIL2 and NEIL3) that showed homology to the Escherichia coli Fpg/Nei DNA glycosylases was identified. Two of the proteins, NEIL1 and NEIL2 have been thoroughly characterized and shown to initiate BER of a diverse number of oxidized lesions. However, much less is known about NEIL3. The biochemical properties of NEIL3 have not been elucidated. This is mainly due to the difficulty in the expression and purification of NEIL3. Here, we describe the expression and partial purification of full-length human NEIL3 and the expression, purification and characterization of a truncated human core-NEIL3 (amino acids 1–301) that contains the complete E. coli Fpg/Nei-like domain but lacks the C-terminal region.  相似文献   

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DNA glycosylases/AP lyases initiate repair of oxidized bases in the genomes of all organisms by excising these lesions and then cleaving the DNA strand at the resulting abasic (AP) sites and generate 3' phospho alpha,beta-unsaturated aldehyde (3' PUA) or 3' phosphate (3' P) terminus. In Escherichia coli, the AP-endonucleases (APEs) hydrolyze both 3' blocking groups (3' PUA and 3' P) to generate the 3'-OH termini needed for repair synthesis. In mammalian cells, the previously characterized DNA glycosylases, NTH1 and OGG1, produce 3' PUA, which is removed by the only AP-endonuclease, APE1. However, APE1 is barely active in removing 3' phosphate generated by the recently discovered mammalian DNA glycosylases NEIL1 and NEIL2. We showed earlier that the 3' phosphate generated by NEIL1 is efficiently removed by polynucleotide kinase (PNK) and not APE1. Here we show that the NEIL2-initiated repair of 5-hydroxyuracil (5-OHU) similarly requires PNK. We have also observed stable interaction between NEIL2 and other BER proteins DNA polymerase beta (Pol beta), DNA ligase IIIalpha (Lig IIIalpha) and XRCC1. In spite of their limited sequence homology, NEIL1 and NEIL2 interact with the same domains of Pol beta and Lig IIIalpha. Surprisingly, while the catalytically dispensable C-terminal region of NEIL1 is the common interacting domain, the essential N-terminal segment of NEIL2 is involved in analogous interaction. The BER proteins including NEIL2, PNK, Pol beta, Lig IIIalpha and XRCC1 (but not APE1) could be isolated as a complex from human cells, competent for repair of 5-OHU in plasmid DNA.  相似文献   

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The repair of free-radical oxidative DNA damage is carried out by lesion-specific DNA glycosylases as the first step of the highly conserved base excision repair (BER) pathway. In humans, three orthologs of the prototypical endonuclease VIII (Nei), the Nei-like NEIL1-3 enzymes are involved in the repair of oxidized DNA lesions. In recent years, several genome and cancer single-nucleotide polymorphic variants of the NEIL1 glycosylase have been identified. In this study we characterized four variants of human NEIL1: S82C, G83D, P208S, and ΔE28, and tested their ability to excise pyrimidine-derived lesions such as thymine glycol (Tg), 5-hydroxyuracil (5-OHU), and dihydrouracil (DHU) and the purine-derived guanidinohydantoin (Gh), spiroiminodihydantoin 1 (Sp1), and methylated 2,6-diamino-4-hydroxy-5-formamidopyrimidine (MeFapyG). The P208S variant has near wild-type activity on all substrates tested. The S82C and ΔE28 variants exhibit decreased Tg excision compared to wild-type. G83D displays little to no activity with any of the substrates tested, with the exception of Gh and Sp1. Human NEIL1 is known to undergo editing whereby the lysine at position 242 is recoded into an arginine. The non-edited form of NEIL1 is more efficient at cleaving Tg than the R242 form, but the G83D variant does not cleave Tg regardless of the edited status of NEIL1. The corresponding G86D variant in Mimivirus Nei1 similarly lacks glycosylase activity. A structure of a G86D–DNA complex reveals a rearrangement in the β4/5 loop comprising Leu84, the highly-conserved void-filling residue, thereby providing a structural rationale for the decreased glycosylase activity of the glycine to aspartate variant.  相似文献   

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Genome instability, primarily caused by faulty DNA repair mechanisms, drives tumorigenesis. Therapeutic interventions that exploit deregulated DNA repair in cancer have made considerable progress by targeting tumor-specific alterations of DNA repair factors, which either induces synthetic lethality or augments the efficacy of conventional chemotherapy and radiotherapy. The study of Fanconi anemia (FA), a rare inherited blood disorder and cancer predisposition syndrome, has been instrumental in understanding the extent to which DNA repair defects contribute to tumorigenesis. The FA pathway functions to resolve blocked replication forks in response to DNA interstrand cross-links (ICLs), and accumulating knowledge of its activation by the ubiquitin-mediated signaling pathway has provided promising therapeutic opportunities for cancer treatment. Here, we discuss recent advances in our understanding of FA pathway regulation and its potential application for designing tailored therapeutics that take advantage of deregulated DNA ICL repair in cancer.  相似文献   

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Mitochondria are membrane-bound organelles found in eukaryotic cells where they generate energy through the respiratory chain. They contain their own genome that encodes genes critical to the mitochondrial function, but most of their protein content is synthetized from nuclear encoded genes. Damages to the mtDNA can cause mutations and rearrangements with an impact on the respiratory functions of the cell. DNA repair factors are able to localize to mitochondria to restore mtDNA integrity and ensure its proper inheritance. We describe in this article the mitochondrial localization of the Mph1/FANCM helicase that serves critical roles in nuclear DNA repair processes. Mph1 localizes to mitochondria and its functions contribute to the mtDNA integrity under mtDNA damaging conditions.  相似文献   

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The SLX4/FANCP tumor suppressor has emerged as a key player in the maintenance of genome stability, making pivotal contributions to the repair of interstrand cross-links, homologous recombination, and in response to replication stress genome-wide as well as at specific loci such as common fragile sites and telomeres. SLX4 does so in part by acting as a scaffold that controls and coordinates the XPF–ERCC1, MUS81–EME1, and SLX1 structure-specific endonucleases in different DNA repair and recombination mechanisms. It also interacts with other important DNA repair and cell cycle control factors including MSH2, PLK1, TRF2, and TOPBP1 as well as with ubiquitin and SUMO. This review aims at providing an up-to-date and comprehensive view on the key functions that SLX4 fulfills to maintain genome stability as well as to highlight and discuss areas of uncertainty and emerging concepts.  相似文献   

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Mammalian AP endonuclease 1 is a pivotal enzyme of the base excision repair pathway acting on apurinic/apyrimidinic sites. Previous structural and biochemical studies showed that the conserved Asn-212 residue is important for the enzymatic activity of APE1. Here, we report a comprehensive pre-steady-state kinetic analysis of two APE1 mutants, each containing amino acid substitutions at position 212, to ascertain the role of Asn-212 in individual steps of the APE1 catalytic mechanism. We applied the stopped-flow technique for detection of conformational transitions in the mutant proteins and DNA substrates during the catalytic cycle, using fluorophores that are sensitive to the micro-environment. Our data indicate that Asn-212 substitution by Asp reduces the rate of the incision step by ∼550-fold, while Ala substitution results in ∼70,000-fold decrease. Analysis of the binding steps revealed that both mutants continued to rapidly and efficiently bind to abasic DNA containing the natural AP site or its tetrahydrofuran analogue (F). Moreover, transient kinetic analysis showed that N212A APE1 possessed a higher binding rate and a higher affinity for specific substrates compared to N212D APE1. Molecular dynamics (MD) simulation revealed a significant dislocation of the key catalytic residues of both mutant proteins relative to wild-type APE1. The analysis of the model structure of N212D APE1 provides evidence for alternate hydrogen bonding between Asn-212 and Asp-210 residues, whereas N212A possesses an extended active site pocket due to Asn removal. Taken together, these biochemical and MD simulation results indicate that Asn-212 is essential for abasic DNA incision, but is not crucial for effective recognition/binding.  相似文献   

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During DNA replication, stalled replication forks and DSBs arise when the replication fork encounters ICLs (interstrand crosslinks), covalent protein/DNA intermediates or other discontinuities in the template. Recently, homologous recombination proteins have been shown to function in replication-coupled repair of ICLs in conjunction with the Fanconi anemia (FA) regulatory factors FANCD2-FANCI, and, conversely, the FA gene products have been shown to play roles in stalled replication fork rescue even in the absence of ICLs, suggesting a broader role for the FA network than previously appreciated. Here we show that DNA2 helicase/nuclease participates in resection during replication-coupled repair of ICLs and other replication fork stresses. DNA2 knockdowns are deficient in HDR (homology-directed repair) and the S phase checkpoint and exhibit genome instability and sensitivity to agents that cause replication stress. DNA2 is partially redundant with EXO1 in these roles. DNA2 interacts with FANCD2, and cisplatin induces FANCD2 ubiquitylation even in the absence of DNA2. DNA2 and EXO1 deficiency leads to ICL sensitivity but does not increase ICL sensitivity in the absence of FANCD2. This is the first demonstration of the redundancy of human resection nucleases in the HDR step in replication-coupled repair, and suggests that DNA2 may represent a new mediator of the interplay between HDR and the FA/BRCA pathway.  相似文献   

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Base excision repair is the major pathway for removal of oxidative DNA base damage. This pathway is initiated by DNA glycosylases, which recognize and excise damaged bases from DNA. In this work, we have purified the glycosylase domain (GD) of human DNA glycosylase NEIL3. The substrate specificity has been characterized and we have elucidated the catalytic mechanisms. GD NEIL3 excised the hydantoin lesions spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh) in single-stranded (ss) and double-stranded (ds) DNA efficiently. NEIL3 also removed 5-hydroxy-2′-deoxycytidine (5OHC) and 5-hydroxy-2′-deoxyuridine (5OHU) in ssDNA, but less efficiently than hydantoins. Unlike NEIL1 and NEIL2, which possess a β,δ-elimination activity, NEIL3 mainly incised damaged DNA by β-elimination. Further, the base excision and strand incision activities of NEIL3 exhibited a non-concerted action, indicating that NEIL3 mainly operate as a monofunctional DNA glycosylase. The site-specific NEIL3 mutant V2P, however, showed a concerted action, suggesting that the N-terminal amino group in Val2 is critical for the monofunctional modus. Finally, we demonstrated that residue Lys81 is essential for catalysis.  相似文献   

14.
Tyrosyl-DNA phosphodiesterase 1 (Tdp1) catalyzes the hydrolysis of the phosphodiester linkage between the DNA 3' phosphate and a tyrosine residue as well as a variety of other DNA 3' damaged termini. Recently we have shown that Tdp1 can liberate the 3' DNA phosphate termini from apurinic/apyrimidinic (AP) sites. Here, we found that Tdp1 is more active in the cleavage of the AP sites inside bubble-DNA structure in comparison to ssDNA containing AP site. Furthermore, Tdp1 hydrolyzes AP sites opposite to bulky fluorescein adduct faster than AP sites located in dsDNA. Whilst the Tdp1 H493R (SCAN1) and H263A mutants retain the ability to bind an AP site-containing DNA, both mutants do not reveal endonuclease activity, further suggesting the specificity of the AP cleavage activity. We suggest that this Tdp1 activity can contribute to the repair of AP sites particularly in DNA structures containing ssDNA region or AP sites in the context of clustered DNA lesions.  相似文献   

15.
During DNA replication, stalled replication forks and DSBs arise when the replication fork encounters ICLs (interstrand crosslinks), covalent protein/DNA intermediates or other discontinuities in the template. Recently, homologous recombination proteins have been shown to function in replication-coupled repair of ICLs in conjunction with the Fanconi anemia (FA) regulatory factors FANCD2-FANCI, and, conversely, the FA gene products have been shown to play roles in stalled replication fork rescue even in the absence of ICLs, suggesting a broader role for the FA network than previously appreciated. Here we show that DNA2 helicase/nuclease participates in resection during replication-coupled repair of ICLs and other replication fork stresses. DNA2 knockdowns are deficient in HDR (homology-directed repair) and the S phase checkpoint and exhibit genome instability and sensitivity to agents that cause replication stress. DNA2 is partially redundant with EXO1 in these roles. DNA2 interacts with FANCD2, and cisplatin induces FANCD2 ubiquitylation even in the absence of DNA2. DNA2 and EXO1 deficiency leads to ICL sensitivity but does not increase ICL sensitivity in the absence of FANCD2. This is the first demonstration of the redundancy of human resection nucleases in the HDR step in replication-coupled repair, and suggests that DNA2 may represent a new mediator of the interplay between HDR and the FA/BRCA pathway.  相似文献   

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Fanconi anemia (FA) is an autosomal or X-linked recessive disorder characterized by chromosomal instability, bone marrow failure, cancer susceptibility, and a profound sensitivity to agents that produce DNA interstrand cross-link (ICL). To date, 15 genes have been identified that, when mutated, result in FA or an FA-like syndrome. It is believed that cellular resistance to DNA interstrand cross-linking agents requires all 15 FA or FA-like proteins. Here, we review our current understanding of how these FA proteins participate in ICL repair and discuss the molecular mechanisms that regulate the FA pathway to maintain genome stability.  相似文献   

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18.
Mgm101 has well-characterized activity for the repair and replication of the mitochondrial genome. Recent work has demonstrated a further role for Mgm101 in nuclear DNA metabolism, contributing to an S-phase specific DNA interstrand cross-link repair pathway that acts redundantly with a pathway controlled by Pso2 exonuclease. Due to involvement of FANCM, FANCJ and FANCP homologues (Mph1, Chl1 and Slx4), this pathway has been described as a Fanconi anemia-like pathway. In this pathway, Mgm101 physically interacts with the DNA helicase Mph1 and the MutSα (Msh2/Msh6) heterodimer, but its precise role is yet to be elucidated. Data presented here suggests that Mgm101 functionally overlaps with Rad52, supporting previous suggestions that, based on protein structure and biochemical properties, Mgm101 and Rad52 belong to a family of proteins with similar function. In addition, our data shows that this overlap extends to the function of both proteins at telomeres, where Mgm101 is required for telomere elongation during chromosome replication in rad52 defective cells. We hypothesize that Mgm101 could, in Rad52-like manner, preferentially bind single-stranded DNAs (such as at stalled replication forks, broken chromosomes and natural chromosome ends), stabilize them and mediate single-strand annealing-like homologous recombination event to prevent them from converting into toxic structures.  相似文献   

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