DNA损伤修复机制——解读2015年诺贝尔化学奖

Tomas Lindahl, Paul Modrich和Aziz Sancar三位科学家因发现“DNA损伤修复机制”获得了2015年诺贝尔化学奖．Lindahl首次发现Escherichia Coli中参与碱基切除修复的第一个蛋白质--尿嘧啶 DNA糖基化酶（UNG）; Modrich重建了错配修复的体外系统,从大肠杆菌到哺乳动物深入探究了错配修复的机制; Sancar利用纯化的UvrA、UvrB、UvrC重建了核苷酸切除修复的关键步骤,阐述了核苷酸切除修复的分子机制．DNA损伤是由生物所处体外环境和体内因素共同导致的,面对不同种类的损伤,机体启动多种不同的修复机制修复损伤,保护基因组稳定性．这些修复机制包括：光修复(light repairing);核苷酸切除修复（nucleotide excision repair, NER）;碱基切除修复（base excision repair, BER）;错配修复（mismatch repair, MMR）;以及DNA双链断裂修复（DNA double strand breaks repair, DSBR）．其中DNA双链断裂修复又分同源重组（homologous recombination, HR）和非同源末端连接（non homologous end joining, NHEJ）两种方式．本文将对上述几种修复的机制进行总结与讨论．     

Removal of N-6-methyladenine by the nucleotide excision repair pathway triggers the repair of mismatches in yeast gap-repair intermediates

Gap-repair assays have been an important tool for studying the genetic control of homologous recombination in yeast. Sequence analysis of recombination products derived when a gapped plasmid is diverged relative to the chromosomal repair template additionally has been used to infer structures of strand-exchange intermediates. In the absence of the canonical mismatch repair pathway, mismatches present in these intermediates are expected to persist and segregate at the next round of DNA replication. In a mismatch repair defective (mlh1Δ) background, however, we have observed that recombination-generated mismatches are often corrected to generate gene conversion or restoration events. In the analyses reported here, the source of the aberrant mismatch removal during gap repair was examined. We find that most mismatch removal is linked to the methylation status of the plasmid used in the gap-repair assay. Whereas more than half of Dam-methylated plasmids had patches of gene conversion and/or restoration interspersed with unrepaired mismatches, mismatch removal was observed in less than 10% of products obtained when un-methylated plasmids were used in transformation experiments. The methylation-linked removal of mismatches in recombination intermediates was due specifically to the nucleotide excision repair pathway, with such mismatch removal being partially counteracted by glycosylases of the base excision repair pathway. These data demonstrate that nucleotide excision repair activity is not limited to bulky, helix-distorting DNA lesions, but also targets removal of very modest perturbations in DNA structure. In addition to its effects on mismatch removal, methylation reduced the overall gap-repair efficiency, but this reduction was not affected by the status of excision repair pathways. Finally, gel purification of DNA prior to transformation reduced gap-repair efficiency four-fold in a nucleotide excision repair-defective background, indicating that the collateral introduction of UV damage can potentially compromise genetic interpretations.     

Rad50/SMC proteins and ABC transporters: unifying concepts from high-resolution structures

ATP-binding cassette (ABC)-type ATPases are chemo-mechanical engines for diverse biological pathways. ABC ATPase domains act not only in ABC transporters but also in DNA mismatch, nucleotide excision and double-strand break repair enzymes, as well as in chromosome segregation. Atomic-resolution crystal structures suggest molecular mechanisms for ABC ATPases and reveal surprisingly significant mechanistic and architectural conservation. This emerging unified structural biochemistry provides general medical and biological insights into how ABC proteins function as chemo-mechanical devices. ATP binding by the signature and Q-loop motifs drives the conformations of substrate-specific domains to accomplish diverse functions in transmembrane transport and DNA repair.     

Nucleotide excision repair and anti-cancer chemotherapy

DNA repair is an important effector of anti-cancer drug resistance. In recent years, it has become apparent that DNA repair is an extremely complex process. Processes within DNA repair that may contribute to one or more drug resistance phenotypes include; O-6-alkyltransferase activity, base excision repair, mismatch repair, nucleotide excision repair, and gene specific repair. Clearly, several of these processes may show increased activity within any single cell, or tumor, at any one time. This review attempts to touch briefly upon the question of the distinctions between each of these specific pathways; and then seeks to expand on nucleotide excision repair as a possible effector of cellular and clinical resistance to platinum-based anticancer therapy. This revised version was published online in August 2006 with corrections to the Cover Date.     

hMutSbeta is required for the recognition and uncoupling of psoralen interstrand cross-links in vitro

The removal of interstrand cross-links (ICLs) from DNA in higher eucaryotes is not well understood. Here, we show that processing of psoralen ICLs in mammalian cell extracts is dependent upon the mismatch repair complex hMutSbeta but is not dependent upon the hMutSalpha complex or hMlh1. The processing of psoralen ICLs is also dependent upon the nucleotide excision repair proteins Ercc1 and Xpf but not upon other components of the excision stage of this pathway or upon Fanconi anemia proteins. Products formed during the in vitro reaction indicated that the ICL has been removed or uncoupled from the cross-linked substrate in the mammalian cell extracts. Finally, the hMutSbeta complex is shown to specifically bind to psoralen ICLs, and this binding is stimulated by the addition of PCNA. Thus, a novel pathway for processing ICLs has been identified in mammalian cells which involves components of the mismatch repair and nucleotide excision repair pathways.     

Nucleotide excision repair deficiency causes elevated levels of chromosome gain in Saccharomyces cerevisiae

Aneuploidy is the most frequent aberration observed in tumor cells, and underlies many debilitating and cancer-prone congenital disorders. Aneuploidy most often arises as a consequence of chromosomal non-disjunction, however, little is known about the genetic and epigenetic factors that affect the chromosomal segregation process. As many cancer-prone syndromes are associated with defects in DNA repair pathways we decided to investigate the relationship between DNA repair in mutation avoidance pathways, namely base and nucleotide excision, and mismatch repair (MMR), and aneuploidy in the yeast Saccharomyces cerevisiae. Isogenic haploid and diploid DNA repair deficient yeast strains were constructed, and spontaneous levels of intra- and inter-chromosomal recombination, forward mutation, chromosome gain, and loss were measured. We show that the nucleotide excision repair (NER) pathway is required for accurate chromosomal disjunction. In the absence of Rad1, Rad2, or Rad4, spontaneous levels of chromosome XV gain were significantly elevated in both haploid and diploid mutant strains. Thus, chromosome gain may be an additional cancer predisposing event in NER deficient patients.     

Short-patch correction of C/C mismatches in human cells

We examined whether the human nucleotide excision repair complex, which is specialized on the removal of bulky DNA adducts, also displays a correcting activity on base mismatches. The cytosine/cytosine (C/C) lesion was used as a model substrate to monitor the correction of base mismatches in human cells. Fibroblasts with different repair capabilities were transfected with shuttle vectors that contain a site-directed C/C mismatch in the replication origin, accompanied by an additional C/C mismatch in one of the flanking sequences that are not essential for replication. Analysis of the vector progeny obtained from these doubly modified substrates revealed that C/C mismatches were eliminated before DNA synthesis not only in the repair-proficient background, but also when the target cells carried a genetic defect in long-patch mismatch repair, in nucleotide excision repair, or when both pathways were deleted. Furthermore, cells deficient for long-patch mismatch repair as well as a cell line that combines mismatch and nucleotide excision repair defects were able to correct multiple C/C mispairs, placed at distances of 21–44 nt, in an independent manner, such that the removal of each lesion led to individual repair patches. These results support the existence of a concurrent short-patch mechanism that rectifies C/C mismatches.     

Evidence for a physical interaction between the Escherichia coli methyl-directed mismatch repair proteins MutL and UvrD.

UvrD (DNA helicase II) is an essential component of two major DNA repair pathways in Escherichia coli: methyl-directed mismatch repair and UvrABC-mediated nucleotide excision repair. In addition, it has an undefined role in the RecF recombination pathway and possibly in replication. In an effort to better understand the role of UvrD in these various aspects of DNA metabolism, a yeast two-hybrid screen was used to search for interacting protein partners. Screening of an E.coli genomic library revealed a potential interaction between UvrD and MutL, a component of the methyl-directed mismatch repair pathway. The interaction was confirmed by affinity chromatography using purified proteins. Deletion analysis demonstrated that the C-terminal 218 amino acids (residues 398-615) of MutL were sufficient to produce the two-hybrid interaction with UvrD. On the other hand, both the N- and C-termini of UvrD were required for interaction with MutL. The implications of this interaction for the mismatch repair mechanism are discussed.     

Nuclear translocation contributes to regulation of DNA excision repair activities

DNA mutations are circumvented by dedicated specialized excision repair systems, such as the base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR) pathways. Although the individual repair pathways have distinct roles in suppressing changes in the nuclear DNA, it is evident that proteins from the different DNA repair pathways interact [Y. Wang, D. Cortez, P. Yazdi, N. Neff, S.J. Elledge, J. Qin, BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures, Genes Dev. 14 (2000) 927–939; M. Christmann, M.T. Tomicic, W.P. Roos, B. Kaina, Mechanisms of human DNA repair: an update, Toxicology 193 (2003) 3–34; N.B. Larsen, M. Rasmussen, L.J. Rasmussen, Nuclear and mitochondrial DNA repair: similar pathways? Mitochondrion 5 (2005) 89–108]. Protein interactions are not only important for function, but also for regulation of nuclear import that is necessary for proper localization of the repair proteins. This review summarizes the current knowledge on nuclear import mechanisms of DNA excision repair proteins and provides a model that categorizes the import by different mechanisms, including classical nuclear import, co-import of proteins, and alternative transport pathways. Most excision repair proteins appear to contain classical NLS sequences directing their nuclear import, however, additional import mechanisms add alternative regulatory levels to protein import, indirectly affecting protein function. Protein co-import appears to be a mechanism employed by the composite repair systems NER and MMR to enhance and regulate nuclear accumulation of repair proteins thereby ensuring faithful DNA repair.     

A novel protein,Rsf1/Pxd1, is critical for the single‐strand annealing pathway of double‐strand break repair in Schizosaccharomyces pombe

The process of single‐strand annealing (SSA) repairs DNA double‐strand breaks that are flanked by direct repeat sequences through the coordinated actions of a series of proteins implicated in recombination, mismatch repair and nucleotide excision repair (NER). Many of the molecular and mechanistic insights gained in SSA repair have principally come from studies in the budding yeast Saccharomyces cerevisiae. However, there is little molecular understanding of the SSA pathway in the fission yeast Schizosaccharomyces pombe. To further our understanding of this important process, we established a new chromosome‐based SSA assay in fission yeast. Our genetic analyses showed that, although many homologous components participate in SSA repair in these species indicating that some evolutionary conservation, Saw1 and Slx4 are not principal agents in the SSA repair pathway in fission yeast. This is in marked contrast to the function of Saw1 and Slx4 in budding yeast. Additionally, a novel genus‐specific protein, Rsf1/Pxd1, physically interacts with Rad16, Swi10 and Saw1 in vitro and in vivo. We find that Rsf1/Pxd1 is not required for NER and demonstrate that, in fission yeast, Rsf1/Pxd1, but not Saw1, plays a critical role in SSA recombination.     

Single‐nucleotide and long‐patch base excision repair of DNA damage in plants

Base excision repair (BER) is a critical pathway in cellular defense against endogenous or exogenous DNA damage. This elaborate multistep process is initiated by DNA glycosylases that excise the damaged base, and continues through the concerted action of additional proteins that finally restore DNA to the unmodified state. BER has been subject to detailed biochemical analysis in bacteria, yeast and animals, mainly through in vitro reproduction of the entire repair reaction in cell‐free extracts. However, an understanding of this repair pathway in plants has consistently lagged behind. We report the extension of BER biochemical analysis to plants, using Arabidopsis cell extracts to monitor repair of DNA base damage in vitro. We have used this system to demonstrate that Arabidopsis cell extracts contain the enzymatic machinery required to completely repair ubiquitous DNA lesions, such as uracil and abasic (AP) sites. Our results reveal that AP sites generated after uracil excision are processed both by AP endonucleases and AP lyases, generating either 5′‐ or 3′‐blocked ends, respectively. We have also found that gap filling and ligation may proceed either through insertion of just one nucleotide (short‐patch BER) or several nucleotides (long‐patch BER). This experimental system should prove useful in the biochemical and genetic dissection of BER in plants, and contribute to provide a broader picture of the evolution and biological relevance of DNA repair pathways.     

The 26S proteasome negatively regulates the level of overall genomic nucleotide excision repair

Regulation of protein expression can be achieved through destruction of proteins by the 26S proteasome. Cellular processes that are regulated by proteolysis include cell cycle progression, stress responses and differentiation. Several nucleotide excision repair proteins in yeast and humans, such as Rad23, Rad4 and XPB, have been shown to co-purify with Cim3 and Cim5, AAA ATPases of the 19S proteasome regulatory subunit. However, it has not been determined if nucleotide excision repair is regulated through protein destruction. We measured nucleotide excision repair in yeast mutants that are defective in proteasome function and found that the repair of the transcribed and non-transcribed strands of an RNA polymerase II-transcribed reporter gene was increased in the absence of proteasome function. Additionally, overexpression of the Rad4 repair protein, which is bound to the repair/proteolytic factor Rad23, conferred higher rates of nucleotide excision repair. Based on our data we suggest that a protein (or proteins) involved in nucleotide excision repair or in regulation of repair is degraded by the 26S proteasome. We propose that decreased proteasome function enables increased DNA repair, due to the transient accumulation of a specific repair factor, perhaps Rad4.     

Methylation Status of Arabidopsis DNA Repair Gene Promoters During Agrobacterium Infection Reveals Epigenetic Changes in Three Generations

Agrobacterium tumefaciens is a unique pathogen with the ability to transfer a portion of its DNA, the T-DNA, to other organisms. The role of DNA repair genes in Agrobacterium transformation remains controversial. In order to understand if the host DNA repair response and dynamics was specific to bacterial factors such as Vir proteins, T-DNA, and oncogenes, we profiled the expression and promoter methylation of various DNA repair genes. These genes belonged to nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR), homologous recombination (HR), and non-homologous end joining (NHEJ) pathways. We infected Arabidopsis plants with different Agrobacterium strains that lacked one or more of the above components so that the influence of the respective factors could be analysed. Our results revealed that the expression and promoter methylation of most DNA repair genes was affected by Agrobacterium, and it was specific to Vir proteins, T-DNA, oncogenes, or the mere presence of bacteria. In order to determine if Agrobacterium induced any transgenerational epigenetic effect on the DNA repair gene promoters, we studied the promoter methylation in two subsequent generations of the infected plants. Promoters of at least three genes, CEN2, RAD51, and LIG4 exhibited transgenerational memory in response to different bacterial factors. We believe that this is the first report of Agrobacterium-induced transgenerational epigenetic memory of DNA repair genes in plants. In addition, we show that Agrobacterium induces short-lived DNA strand breaks in Arabidopsis cells, irrespective of the presence or absence of virulence genes and T-DNA.

     

DNA base repair – recognition and initiation of catalysis

Endogenous DNA damage induced by hydrolysis, reactive oxygen species and alkylation modifies DNA bases and the structure of the DNA duplex. Numerous mechanisms have evolved to protect cells from these deleterious effects. Base excision repair is the major pathway for removing base lesions. However, several mechanisms of direct base damage reversal, involving enzymes such as transferases, photolyases and oxidative demethylases, are specialized to remove certain types of photoproducts and alkylated bases. Mismatch excision repair corrects for misincorporation of bases by replicative DNA polymerases. The determination of the 3D structure and visualization of DNA repair proteins and their interactions with damaged DNA have considerably aided our understanding of the molecular basis for DNA base lesion repair and genome stability. Here, we review the structural biochemistry of base lesion recognition and initiation of one-step direct reversal (DR) of damage as well as the multistep pathways of base excision repair (BER), nucleotide incision repair (NIR) and mismatch repair (MMR).     

Five repair pathways in one context: chromatin modification during DNA repair.

The eukaryotic cell is faced with more than 10 000 various kinds of DNA lesions per day. Failure to repair such lesions can lead to mutations, genomic instability, or cell death. Therefore, cells have developed 5 major repair pathways in which different kinds of DNA damage can be detected and repaired: homologous recombination, nonhomologous end joining, nucleotide excision repair, base excision repair, and mismatch repair. However, the efficient repair of DNA damage is complicated by the fact that the genomic DNA is packaged through histone and nonhistone proteins into chromatin, a highly condensed structure that hinders DNA accessibility and its subsequent repair. Therefore, the cellular repair machinery has to circumvent this natural barrier to gain access to the damaged site in a timely manner. Repair of DNA lesions in the context of chromatin occurs with the assistance of ATP-dependent chromatin-remodeling enzymes and histone-modifying enzymes, which allow access of the necessary repair factors to the lesion. Here we review recent studies that elucidate the interplay between chromatin modifiers / remodelers and the major DNA repair pathways.     

DNA repair defects in colon cancer

Defects in DNA-repair pathways lead to an accumulation of mutations in genomic DNA that result from non-repair or mis-repair of modifications introduced into the DNA by endogenous or exogenous agents or by the malfunction of DNA metabolic pathways. Until recently, only two repair pathways, postreplicative mismatch repair and nucleotide excision repair, have been linked to cancer in mammals, but these have been joined in recent months also by the damage-reversal and base-excision-repair processes, which have been shown to be inactivated, either through mutation or epigenetically, in human cancer.     

Genomic approaches to DNA repair and mutagenesis

DNA damage is a constant threat to cells, causing cytotoxicity as well as inducing genetic alterations. The steady-state abundance of DNA lesions in a cell is minimized by a variety of DNA repair mechanisms, including DNA strand break repair, mismatch repair, nucleotide excision repair, base excision repair, and ribonucleotide excision repair. The efficiencies and mechanisms by which these pathways remove damage from chromosomes have been primarily characterized by investigating the processing of lesions at defined genomic loci, among bulk genomic DNA, on episomal DNA constructs, or using in vitro substrates. However, the structure of a chromosome is heterogeneous, consisting of heavily protein-bound heterochromatic regions, open regulatory regions, actively transcribed genes, and even areas of transient single stranded DNA. Consequently, DNA repair pathways function in a much more diverse set of chromosomal contexts than can be readily assessed using previous methods. Recent efforts to develop whole genome maps of DNA damage, repair processes, and even mutations promise to greatly expand our understanding of DNA repair and mutagenesis. Here we review the current efforts to utilize whole genome maps of DNA damage and mutation to understand how different chromosomal contexts affect DNA excision repair pathways.     

DNA ligation during excision repair in yeast cell-free extracts is specifically catalyzed by the CDC9 gene product

Excision repair of DNA is an important cellular response to DNA damage induced by radiation and many chemicals. In eukaryotes, base excision repair (BER) and nucleotide excision repair (NER) are two major excision repair pathways which are completed by a DNA ligation step. Using a cell-free system, we have determined the DNA ligase requirement during BER and NER of the yeast S. cerevisiae. Under nonpermissive conditions in extracts of the cdc9-2 temperature-sensitive mutant, DNA ligation in both BER and NER pathways was defective, and the repair patches were enlarged. At the permissive temperature (23 degrees C), DNA ligation during excision repair was only partially functional in the mutant extracts. In contrast, deleting the DNA ligase IV gene did not affect DNA ligation of BER or NER. Defective DNA ligation of BER and NER in cdc9-2 mutant extracts was complemented in vitro by purified yeast Cdc9 protein, but not by DNA ligase IV even when overexpressed. These results demonstrate that the ligation step of excision repair in yeast cell-free extracts is catalyzed specifically by the Cdc9 protein, the homologue of mammalian DNA ligase I.     

Regulation of the DNA replication fork: a way to fight genomic instability

DNA replication is a complex mechanism that functions due to the coordinated interplay of many factors. In the last few years, numerous studies have suggested that DNA replication factors are closely implicated in several DNA transaction events that maintain the integrity of the genome. Therefore, DNA replication fork factors have to be considered as part of a general process that aims to protect and replicate the genome in order to allow correct functioning of a cell and its eventual daughter cells. This is illustrated by the numerous factors that have a well-defined function at the DNA replication fork, but also play crucial roles in different DNA repair pathways such as base excision repair, nucleotide excision repair, double-strand break repair, and mismatch repair. Moreover, several of the replisome proteins have also been shown to be essential in sensing and transducing DNA damages through the checkpoint cascade pathways, including the recently characterised alternative clamps and clamp-loaders. In this review we present DNA replication factors that are involved in different DNA transaction and checkpoint regulation pathways, with emphasis on the link between DNA replication and maintenance of genomic stability.