Reconstitution of 5'-directed human mismatch repair in a purified system

This paper reports reconstitution of 5'-nick-directed mismatch repair using purified human proteins. The reconstituted system includes MutSalpha or MutSbeta, MutLalpha, RPA, EXO1, HMGB1, PCNA, RFC, polymerase delta, and ligase I. In this system, MutSbeta plays a limited role in repair of base-base mismatches, but it processes insertion/deletion mispairs much more efficiently than MutSalpha, which efficiently corrects both types of heteroduplexes. MutLalpha reduces the processivity of EXO1 and terminates EXO1-catalyzed excision upon mismatch removal. In the absence of MutLalpha, mismatch-provoked excision by EXO1 occurs extensively. RPA and HMGB1 play similar but complementary roles in stimulating MutSalpha-activated, EXO1-catalyzed excision in the presence of a mismatch, but RPA has a distinct role in facilitating MutLalpha-mediated excision termination past mismatch. Evidence is provided that efficient repair of a single mismatch requires multiple molecules of MutSalpha-MutLalpha complex. These data suggest a model for human mismatch repair involving coordinated initiation and termination of mismatch-provoked excision.     

Differential requirement for proliferating cell nuclear antigen in 5' and 3' nick-directed excision in human mismatch repair

Proliferating cell nuclear antigen (PCNA) is involved in mammalian mismatch repair at a step prior to or at mismatch excision, but the molecular mechanism of this process is not fully understood. To examine the role of PCNA in mismatch-provoked and nick-directed excision, orientation-specific mismatch removal of heteroduplexes with a pre-existing nick was monitored in human nuclear extracts supplemented with the PCNA inhibitor protein p21. We show here that, whereas 3' nick-directed mismatch excision was completely inhibited by low concentrations of p21 or a p21 C-terminal fusion protein, 5' nick-directed excision was only partially blocked under the same conditions. No further reduction of the 5' excision was detected when a much higher concentration of p21 C-terminal protein was used. These results suggest the following. (i) There is a differential requirement for PCNA in 3' and 5' nick-directed excision; and (ii) 5' nick-directed excision is conducted by a manner either dependent on or independent of PCNA. Our in vitro reconstitution experiments indeed identified a 5' nick-directed excision pathway that is dependent on PCNA, hMutSalpha, and a partially purified fraction from a HeLa nuclear extract.     

Recognition and repair of compound DNA lesions (base damage and mismatch) by human mismatch repair and excision repair systems.

Nucleotide excision repair and the long-patch mismatch repair systems correct abnormal DNA structures arising from DNA damage and replication errors, respectively. DNA synthesis past a damaged base (translesion replication) often causes misincorporation at the lesion site. In addition, mismatches are hot spots for DNA damage because of increased susceptibility of unpaired bases to chemical modification. We call such a DNA lesion, that is, a base damage superimposed on a mismatch, a compound lesion. To learn about the processing of compound lesions by human cells, synthetic compound lesions containing UV photoproducts or cisplatin 1,2-d(GpG) intrastrand cross-link and mismatch were tested for binding to the human mismatch recognition complex hMutS alpha and for excision by the human excision nuclease. No functional overlap between excision repair and mismatch repair was observed. The presence of a thymine dimer or a cisplatin diadduct in the context of a G-T mismatch reduced the affinity of hMutS alpha for the mismatch. In contrast, the damaged bases in these compound lesions were excised three- to fourfold faster than simple lesions by the human excision nuclease, regardless of the presence of hMutS alpha in the reaction. These results provide a new perspective on how excision repair, a cellular defense system for maintaining genomic integrity, can fix mutations under certain circumstances.     

PARP-1 enhances the mismatch-dependence of 5'-directed excision in human mismatch repair in vitro

End-directed mismatch-provoked excision has been reconstituted in several purified systems. While 3'-directed excision displays a mismatch dependence similar to that observed in nuclear extracts (≈20-fold), the mismatch dependence of 5'-directed excision is only 3-4-fold, significantly less than that in extracts (8-10-fold). Utilizing a fractionation-based approach, we have isolated a single polypeptide that enhances mismatch dependence of reconstituted 5'-directed excision and have shown it to be identical to poly[ADP-ribose] polymerase 1 (PARP-1). Titration of reconstituted excision reactions or PARP-1-depleted HeLa nuclear extract with purified PARP-1 showed that the protein specifically enhances mismatch dependence of 5'-directed excision. Analysis of a set of PARP-1 mutants revealed that the DNA binding domain and BRCT fold contribute to the regulation of excision specificity. Involvement of the catalytic domain is restricted to its ability to poly(ADP-ribosyl)ate PARP-1 in the presence of NAD(+), likely through interference with DNA binding. Analysis of protein-protein interactions demonstrated that PARP-1 interacts with mismatch repair proteins MutSα, exonuclease 1, replication protein A (RPA), and as previously shown by others, replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) as well. The BRCT fold plays an important role in the interaction of PARP-1 with the former three proteins.     

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.     

Mismatch repair in human nuclear extracts: effects of internal DNA-hairpin structures between mismatches and excision-initiation nicks on mismatch correction and mismatch-provoked excision

DNA mismatch repair (MMR) couples recognition of base mispairs by MSH2.MSH6 heterodimers to initiation, hundreds of nucleotides away, of nascent strand 3'-5' or 5'-3' excision through the mispair. Mismatch-recognition complexes have been hypothesized to move along DNA to excision-initiation signals, in eukaryotes, perhaps ends of nascent DNA, or to remain at mismatches and search through space for initiation signals. Subsequent MMR excision, whether simple processive digestion of the targeted strand or tracking of an excision complex, remains poorly understood. In human cell-free extracts, we analyzed correction of a mismatch in a 2.2-kilobase pair (kbp) circular plasmid containing a pre-existing excision-initiation nick for initiation, and measured MMR excision (in the absence of exogenous dNTPs) at specific locations. Excision specificities were approximately 100:1 for nicked versus continuous strands, 80:1 for mismatched versus homoduplex DNA, and 30:1 for shorter (0.3-kbp) versus longer (1.9-kbp) nick-mispair paths. To test models for recognition-excision coupling and excision progress, we inserted potential blockades, 20-bp hairpins, into nick-mispair paths, using a novel technique to first generate gapped plasmid. Continuous strand longer-path hairpins did not affect mismatch correction, but shorter-path hairpins reduced correction 4-fold, and both together eliminated it. Shorter-path hairpins had little effect on initiation of (3'-5') excision, measured 30-60 nucleotides 5' to the nick, but blocked subsequent progress of excision to the mismatch; longer-path hairpins blocked the (lower level) 5'-3' excision to the mismatch. Thus, (a) MMR excision protein(s) cannot move past DNA hairpins. Hairpins at both ends of substrate-derived 0.5-kbp DNA fragments did not prevent ATP-induced dissociation of mismatch-bound human MSH2.MSH6, so recognition complexes at mismatches might provoke excision at nicks beyond hairpins, or loosely sliding MSH2.MSH6 dimers might move to the nicks.     

Mismatch recognition and uracil excision provide complementary paths to both Ig switching and the A/T-focused phase of somatic mutation

AID-mediated deamination of dC residues within the immunoglobulin locus generates dU:dG lesions whose resolution leads to class-switch recombination and somatic hypermutation. The dU:dG pair is a mismatch and comprises a base foreign to DNA and is, thus, recognized by proteins from both base excision (uracil-DNA glycosylase, UNG) and mismatch recognition (MSH2/MSH6) pathways. Strikingly, while antibody diversification is perturbed by single deficiency in either UNG or MSH2, combined UNG/MSH2 deficiency leads to a total ablation both of switch recombination and of IgV hypermutation at dA:dT pairs. The initiating dU:dG lesions appear not to be recognized and are simply replicated over. The results indicate that the major pathway for switch recombination occurs through uracil excision with mismatch recognition of dU:dG providing a backup; the second phase of hypermutation (essentially introducing mutations solely at dA:dT pairs) is triggered by mismatch recognition of the dU:dG lesion with uracil excision providing a backup.     

Mechanism of 5'-directed excision in human mismatch repair

We have developed a purified system that supports mismatch-dependent 5'-->3' excision. In the presence of RPA, ATP, and a mismatch, MutSalpha activates 5'-->3' excision by EXOI, and excision terminates after removal of the mispair. MutSalpha confers high processivity on EXOI, and termination is due to RPA-dependent displacement of this processive complex from the helix and a weak ability of EXOI to reload at the RPA-bound gap in the product, as well as MutSalpha- and MutLalpha-dependent suppression of EXOI activity in the absence of a mismatch cofactor. As observed in the purified system, excision directed by a 5' strand break in HeLa nuclear extract can proceed in the absence of MutLalpha or PCNA, although 3' excision in the extract system requires both proteins.     

Signaling from DNA mispairs to mismatch-repair excision sites despite intervening blockades

Mismatch-repair (MMR) systems promote genomic stability by correction of DNA replication errors. Thus, MMR proteins--prokaryotic MutS and MutL homodimers or their MutSalpha and MutLalpha heterodimer homologs, plus accessory proteins--specifically couple mismatch recognition to nascent-DNA excision. In vivo excision-initiation signals--specific nicks in some prokaryotes, perhaps growing 3' ends or Okazaki-fragment 5' ends in eukaryotes--are efficiently mimicked in vitro by nicks or gaps in exogenous DNA substrates. In some models for recognition-excision coupling, MutSalpha bound to mismatches is induced by ATP hydrolysis, or simply by binding of ATP, to slide along DNA to excision-initiation sites, perhaps in association with MutLalpha and accessory proteins. In other models, MutSalpha.MutLalpha complexes remain fixed at mismatches and contact distant excision sites by DNA looping. To challenge the hypothesis that recognition complexes remain fixed, we placed biotin-streptavidin blockades between mismatches and pre-existing nicks. In human nuclear extracts, mismatch efficiently provoked the initiation of excision despite the intervening barriers, as predicted. However, excision progress and therefore mismatch correction were prevented.     

Mismatch excision and possible polarity effects result in preferred deoxyribonucleic acid strand of integration in pneumococcal transformation.

Heteroduplex deoxyribonucleic acid molecules having a drug resistance marker on one strand and its wild-type allele on the other have been used as donors in pneumococcal transformation. Opposite strands are not equally effective in producing transformants, and this strand bias is not the same, either in direction or magnitude, for various different genetic markers. Selective excision of mismatched base pairs is probably responsible for the large differences in strand efficiency seen with discriminating (hex+) strains, for when the recipient is nondiscriminating (hex-), and therefore presumably lacking an excision enzyme system, strand bias is drastically reduced or altered. The evidence also indicates that excision occurs after integration, as it is provoked by specific donor-recipient mismatch and not by the same mismatch when introduced within donor heteroduplex molecules. Excision can extend to include a neighboring linked marker which would otherwise not be excised, thus altering its intrinsic strand bias as well as its efficiency in transformation. There is a small bias in relative strand efficiency for some markers, not caused by mismatch excision, which perhaps is due to polarity in the integration process itself.     

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.     

Initiation of repair of A/G mismatches is modulated by sequence context

The efficiency of DNA glycosylases to initiate base excision repair (BER) has been demonstrated to be modulated by the precise sequence context in which the lesion or mismatch is located. In the case of DNA containing an A/G mismatch, in which the recognition and excision of adenine from the mismatch is mediated by the Escherichia coli MutY enzyme, not only does the local sequence context affect the strength of base stacking interactions, but it also modulates the syn/anti conformation around the glycosyl bond of the bases in the mispair. Utilizing prior NMR data to identify DNA sequence contexts that adopt either an anti/anti or a syn/anti configuration at an A/G mismatch, we tested the hypothesis that the initial equilibrium of the mismatched base orientations would modulate the overall efficiency of glycosyl bond scission. By systematically varying the sequence context around a central A/G mismatch within a 30-mer duplex DNA, significant kinetic differences were observed that were consistent with this hypothesis. Since the relative efficiency of the kinetics fell into only two groupings, a NMR study was conducted on a DNA sequence context of unknown syn/anti conformation. These data established that the relative syn/anti conformation did not correlate with the excision efficiency, as well as there being a lack of correlation between kinetics and thermal stability of these DNAs.     

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.     

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．     

Mispair-bound human MutS–MutL complex triggers DNA incisions and activates mismatch repair

DNA mismatch repair (MMR) relies on MutS and MutL ATPases for mismatch recognition and strand-specific nuclease recruitment to remove mispaired bases in daughter strands. However, whether the MutS–MutL complex coordinates MMR by ATP-dependent sliding on DNA or protein–protein interactions between the mismatch and strand discrimination signal is ambiguous. Using functional MMR assays and systems preventing proteins from sliding, we show that sliding of human MutSα is required not for MMR initiation, but for final mismatch removal. MutSα recruits MutLα to form a mismatch-bound complex, which initiates MMR by nicking the daughter strand 5′ to the mismatch. Exonuclease 1 (Exo1) is then recruited to the nick and conducts 5′ → 3′ excision. ATP-dependent MutSα dissociation from the mismatch is necessary for Exo1 to remove the mispaired base when the excision reaches the mismatch. Therefore, our study has resolved a long-standing puzzle, and provided new insights into the mechanism of MMR initiation and mispair removal.Subject terms: Molecular biology     

Functional mutation of DNA polymerase beta found in human gastric cancer--inability of the base excision repair in vitro.

DNA polymerase beta (polbeta) is one of mammalian DNA polymerases and is known to be involved in a G:T/G:U mismatch repair. In order to investigate an involvement of this enzyme in a base excision repair, we searched a mutation of human polbeta in human gastric cancer and studied a function of the mutation. We observed cancer-specific missense mutations in 6 of 20 samples. All of these mutations were, however, heterozygous. We further analyzed the base excision repair activity of these mutants to know whether these mutants cause an error of mismatch repair. One of these mutants, which resulted in an amino acid substitution of Glu for Lys at codon 295, showed an inhibitory effect by in vitro base excision repair assay, suggesting that this mutation might play some role in carcinogenesis of the gastric mucosa.     

Mismatch Repair Mutations of ESCHERICHIA COLI K12 Enhance Transposon Excision

Excision of the prokaryotic transposon Tn10 is a host-mediated process that occurs in the absence of recA function or any transposon-encoded functions. To determine which host functions might play a role in transposon excision, we have isolated 40 mutants of E. coli K12, designated tex, which increase the frequency of Tn10 precise excision. Three of these mutations (texA) have been shown to qualitatively alter RecBC function. We show that 21 additional tex mutations with a mutator phenotype map to five genes previously identified as components of a methylation-directed pathway for repair of base pair mismatches: uvrD, mutH, mutL, mutS and dam. Previously identified alleles of these genes also have a Tex phenotype.--Several other E. coli mutations affecting related functions have been analyzed for their effects on Tn10 excision. Other mutations affecting the frequency of spontaneous mutations (mutT, polA, ung), different excision repair pathways (uvrA, uvrB) or the state of DNA methylation (dcm) have no effect on Tn10 excision. Mutations ssb-113 and mutD5, however, do increase Tn10 excision.--The products of the mismatch correction genes probably function in a coordinated way during DNA repair in vivo. Thus, mutations in these genes might also enhance transposon excision by a single general mechanism. Alternatively, since mutations in each gene have qualitatively and quantitatively different effects on transposon excision, defects in different mismatch repair genes may enhance excision by different mechanisms.     

In vitro studies of DNA mismatch repair proteins

The ability to monitor and characterize DNA mismatch repair activity in various mammalian cells is important for understanding mechanisms involved in mutagenesis and tumorigenesis. Since mismatch repair proteins recognize mismatches containing both normal and chemically altered or damaged bases, in vitro assays must accommodate a variety of mismatches in different sequence contexts. Here we describe the construction of DNA mismatch substrates containing G:T or O⁶meG:T mismatches, the purification of recombinant native human MutSα (MSH2–MSH6) and MutLα (MLH1–PMS2) proteins, and in vitro mismatch repair and excision assays that can be adapted to study mismatch repair in nuclear extracts from mismatch repair proficient and deficient cells.     

Characterization of palindromic loop mismatch repair tracts in mammalian cells

Single- and multi-base (loop) mismatches can arise in DNA by replication errors, during recombination, and by chemical modification of DNA. Single-base and loop mismatches of several nucleotides are efficiently repaired in mammalian cells by a nick-directed, MSH2-dependent mechanism. Larger loop mismatches (> or =12 bases) are repaired by an MSH2-independent mechanism. Prior studies have shown that 12- and 14-base palindromic loops are repaired with bias toward loop retention, and that repair bias is eliminated when five single-base mismatches flank the loop mismatch. Here we show that one single-base mismatch near a 12-base palindromic loop is sufficient to eliminate loop repair bias in wild-type, but not MSH2-defective mammalian cells. We also show that palindromic loop and single-base mismatches separated by 12 bases are repaired independently at least 10% of the time in wild-type cells, and at least 30% of the time in MSH2-defective cells. Palindromic loop and single-base mismatches separated by two bases were never repaired independently. These and other data indicate that loop repair tracts are variable in length. All tracts extend at least 2 bases, some extend <12 bases, and others >12 bases, on one side of the loop. These properties distinguish palindromic loop mismatch repair from the three known excision repair pathways: base excision repair which has one to six base tracts, nucleotide excision repair which has approximately 30 base tracts, and MSH2-dependent mismatch repair, which has tracts that extend for several hundred bases.