DNA错配修复蛋白MutS和MutL的相互作用研究

MutL 和 MutS 是DNA错配修复系统中起关键作用的修复蛋白. 利用基因融合技术高效表达了MutL 和 MutS融合蛋白,并利用它们发展了一种研究二者相互作用的简便方法. 融合蛋白MutL-GFP (Trx-His6-GFP-(Ser-Gly)6-MutL),MutL-Strep tagⅡ (Trx-His6-(Ser-Gly)6-Strep tagⅡ-(Ser-Gly)6-MutL) 和 MutS (Trx-His6-(Ser-Gly)6-MutS) 被构建并在大肠杆菌中高效表达. 收集菌体细胞、超声波破碎后离心取上清进行SDS-聚丙烯酰胺凝胶电泳 (SDS-PAGE) 分析,结果表明有与预期分子质量相应的诱导表达条带出现,其表达量约占全细胞蛋白的30％且以可溶形式存在. 利用固定化金属离子配体亲和层析柱分别纯化融合蛋白,其纯度达到90％. 通过将MutS蛋白固定的方法研究两种MutL融合蛋白分别与MutS之间的相互作用. 结果表明：只有MutS蛋白与含有错配碱基DNA分子结合后才与MutL蛋白发生相互作用. 通过检测MutL融合蛋白标记的绿色荧光信号或酶学显色信号来鉴定相互作用的发生. 建立的融合分子系统方法也为研究其他的蛋白质或生物大分子之间的相互作用提供了一个技术平台.     

Histone Deacetylase 10 Regulates DNA Mismatch Repair and May Involve the Deacetylation of MutS Homolog 2

MutS homolog 2 (MSH2) is an essential DNA mismatch repair (MMR) protein. It interacts with MSH6 or MSH3 to form the MutSα or MutSβ complex, respectively, which recognize base-base mispairs and insertions/deletions and initiate the repair process. Mutation or dysregulation of MSH2 causes genomic instability that can lead to cancer. MSH2 is acetylated at its C terminus, and histone deacetylase (HDAC6) deacetylates MSH2. However, whether other regions of MSH2 can be acetylated and whether other histone deacetylases (HDACs) and histone acetyltransferases (HATs) are involved in MSH2 deacetylation/acetylation is unknown. Here, we report that MSH2 can be acetylated at Lys-73 near the N terminus. Lys-73 is highly conserved across many species. Although several Class I and II HDACs interact with MSH2, HDAC10 is the major enzyme that deacetylates MSH2 at Lys-73. Histone acetyltransferase HBO1 might acetylate this residue. HDAC10 overexpression in HeLa cells stimulates cellular DNA MMR activity, whereas HDAC10 knockdown decreases DNA MMR activity. Thus, our study identifies an HDAC10-mediated regulatory mechanism controlling the DNA mismatch repair function of MSH2.     

Slow Conformational Changes in MutS and DNA Direct Ordered Transitions between Mismatch Search,Recognition and Signaling of DNA Repair

MutS functions in mismatch repair (MMR) to scan DNA for errors, identify a target site and trigger subsequent events in the pathway leading to error removal and DNA re-synthesis. These actions, enabled by the ATPase activity of MutS, are now beginning to be analyzed from the perspective of the protein itself. This study provides the first ensemble transient kinetic data on MutS conformational dynamics as it works with DNA and ATP in MMR. Using a combination of fluorescence probes (on Thermus aquaticus MutS and DNA) and signals (intensity, anisotropy and resonance energy transfer), we have monitored the timing of key conformational changes in MutS that are coupled to mismatch binding and recognition, ATP binding and hydrolysis, as well as sliding clamp formation and signaling of repair. Significant findings include (a) a slow step that follows weak initial interaction between MutS and DNA, in which concerted conformational changes in both macromolecules control mismatch recognition, and (b) rapid, binary switching of MutS conformations that is concerted with ATP binding and hydrolysis and (c) is stalled after mismatch recognition to control formation of the ATP-bound MutS sliding clamp. These rate-limiting pre- and post-mismatch recognition events outline the mechanism of action of MutS on DNA during initiation of MMR.     

Functional Specifics of the MutL Protein of the DNA Mismatch Repair System in Different Organisms

Russian Journal of Bioorganic Chemistry - A DNA mismatch repair (MMR) system is found in all living organisms. MMR dysfunction at any step of DNA repair leads to an accumulation of mutations in the...     

DNA错配修复与肺癌

肺癌是目前世界上最常见的恶性肿瘤之一,虽然近年来对其研究较多,但其发生发展的确切机制仍不清楚。DNA错配修复作为一种重要的复制后修复系统,在确保DNA复制保真性、控制基因突变和维持基因组稳定等方面具有重要作用。近年研究表明,DNA错配修复系统与肺癌的发生、治疗及预后判断有着密切关系。本文主要对DNA错配修复系统在肺癌中的研究进展作一简要综述。     

MutL Protein from the Neisseria gonorrhoeae Mismatch Repair System: Interaction with ATP and DNA

Molecular Biology - The mismatch repair system (MMR) ensures the stability of genetic information during DNA replication in almost all organisms. Mismatch repair is initiated after recognition of a...     

新发现的人类错配DNA修复蛋白0hMLH3

近年来的研究认为 ,防止人类ＤＮＡ复制错误的因素涉及 5个错配修复 (ＭＭＲ)蛋白 ,即ｈＭＳＨ2、ｈＭＳＨ3、ｈＭＳＨ6、ｈＭＬＨ1和ｈＰＭＳ2。最新研究报告发现了ＭＭＲ家族的第 6种成员 ,ｈＭＬＨ3,也与错配修复过程有关[1] 。　　错配修复是将ＤＮＡ复制时产生的错误修复 ,是维持正确的遗传信息的重要机制。错配修复机制的异常是遗传倾向性癌症产生的原因之一。人的错配修复过程与大肠杆菌类似。都有几种蛋白质的复合物的参与。即有错配结合活性的ＭｕｔＳ同源二聚体和蛋白质相互作用的ＭｕｔＬ同源二聚体 ,这两种同源二聚体又结合形成…     

Evolutionary Consequences of DNA Mismatch Inhibited Repair Opportunity

Double strand breaks (DSBs) are often repaired via homologous recombination. Recombinational repair processes are expected to be influenced by nucleotide heterozygosity through mismatch detection systems. Unrepaired DSBs have severe biological consequences and are often lethal. We show that natural selection due to inhibition of recombinational repair associated with polymorphisms could influence their molecular evolution. The main conclusions from this analysis are that, for increasing population size, mismatch detection leads to a limit on average heterozygosity of otherwise selectively neutral polymorphism, an excess of rare variants, and a slowing down of the rate of neutral molecular evolution. The first two results suggest that mismatch detection may account for the surprisingly narrow range of observed average heterozygosities, given the great variation in population size between species.     

DNA错配修复系统研究进展

DNA错配修复(mismatch repair, MMR)系统广泛存在于生物体中.从原核生物大肠杆菌到真核生物及人类,MMR系统有不同的组成成分和修复机制.人体内MMR基因缺陷会造成基因组的不稳定并诱发遗传性非息肉型直肠癌以及其他自发性肿瘤.大肠杆菌MMR系统中的MutS蛋白可特异识别错配或未配对碱基,目前已经发展了多种基于MutS蛋白的基因突变/多态性检测技术.     

Proteomic Analysis Reveals a Novel Mutator S (MutS) Partner Involved in Mismatch Repair Pathway

The mismatch repair (MMR) family is a highly conserved group of proteins that function in correcting base–base and insertion–deletion mismatches generated during DNA replication. Disruption of this process results in characteristic microsatellite instability (MSI), repair defects, and susceptibility to cancer. However, a significant fraction of MSI-positive cancers express MMR genes at normal levels and do not carry detectable mutation in known MMR genes, suggesting that additional factors and/or mechanisms may exist to explain these MSI phenotypes in patients. To systematically investigate the MMR pathway, we conducted a proteomic analysis and identified MMR-associated protein complexes using tandem-affinity purification coupled with mass spectrometry (TAP-MS) method. The mass spectrometry data have been deposited to the ProteomeXchange with identifier PXD003014 and DOI 10.6019/PXD003014. We identified 230 high-confidence candidate interaction proteins (HCIPs). We subsequently focused on MSH2, an essential component of the MMR pathway and uncovered a novel MSH2-binding partner, WDHD1. We further demonstrated that WDHD1 forms a stable complex with MSH2 and MSH3 or MSH6, i.e. the MutS complexes. The specific MSH2/WDHD1 interaction is mediated by the second lever domain of MSH2 and Ala¹¹²³ site of WDHD1. Moreover, we showed that, just like MSH2-deficient cells, depletion of WDHD1 also led to 6-thioguanine (6-TG) resistance, indicating that WDHD1 likely contributes to the MMR pathway. Taken together, our study uncovers new components involved in the MMR pathway, which provides candidate genes that may be responsible for the development of MSI-positive cancers.Cells are equipped with a number of repair mechanisms to correct various types of DNA lesions. At least five major complimentary, but partially overlapping, multistep damage repair pathways are known to operate in mammals: mismatch repair (MMR)¹, nucleotide excision repair, base excision repair, and double-strand break repair, which includes both homologous recombination repair and nonhomologous end joining (see review: (1, 2)). In particular, MMR is a major repair pathway that prevents both base substitution and insertion–deletion mismatches due to replication errors (3–5).MMR is a highly conserved biological pathway that exists from bacteria to mammals. MMR process can be divided into three key steps: mismatch recognition, excision, and resynthesis (5, 6). The initial mismatch recognition step is fulfilled by MutS protein complexes, either MutSα (the MSH2-MSH6 heterodimer) or MutSβ (the MSH2-MSH3 heterodimer). The MutSα is primarily responsible for repairing base–base mismatches and small insertion–deletion loops of 1–2 nucleotides (7–9), while MutSβ preferentially recognizes larger insertion–deletion loops containing up to 14 extra nucleotides (10–12). Binding to mispaired DNA primes MutS to undergo a conformational change and recruitment of MutL to form an ATP-dependent ternary complex (13). Three different MutL heterodimeric complexes, MutLα, MutLβ, and MutLγ have been identified in the mammalian system. MLH1 heterodimerizes with PMS2, PMS1, or MLH3 to form MutLα, MutLβ, or MutLγ, respectively. MutLα plays a crucial role in MMR, as cells that lack either protein inactivate MMR in human cells, while loss of MutLβ or MutLγ heterodimers leads to minor defects in MMR. MutL is able to recognize and excise the lagging strand from the mismatch both distally and proximally (14, 15). Moreover, MutL interacts physically with MutS, enhances mismatch recognition, and recruits and activates exonuclease1 (EXO1) (16, 17). Exonuclease1 (EXO1) is the only enzyme with capabilities to excise nucleotide in 5′-3′ direction (18). In the case of 3′ excision, proliferating cell nuclear antigen (PCNA)/replication factor C-dependent endonuclease activity plays a critical role in 3′-5′ excision involving EXO1. EXO1 then excises nascent DNA from the nick toward and beyond the mismatch to generate a single-strand gap, which is filled by DNA polymerases δ (lagging strand) or ε (leading strand) using the parental DNA strand as a template. Finally, the nick is sealed by DNA ligase I (19, 20). In addition, two MutS homologues, MSH4 and MSH5, share similar structure and sequence features with the other members of the MutS family. Recent evidence suggests that they function beyond MMR and are involved in processes such as recombinant repair, DNA damage signaling, and immunoglobulin class switch recombination (21, 22).It has been well documented that impairment of MMR genes, especially MSH2 and MLH1, cause susceptibility to certain types of cancer, including human nonpolyposis colorectal cancer. At the cellular level, deficient MMR results in a strong mutator phenotype known as microsatellite instability (MSI), which is a hallmark of MMR deficiency (3–5). However, a significant fraction of MSI-positive colorectal cancers express MMR genes at normal levels and do not carry detectable mutation or hypermethylation in known MMR genes (23). Similarly, certain noncolorectal cancer cells with MSI also appear to have normal expression of known MMR protein (24, 25). These observations suggest that additional factors and/or mechanisms may exist to explain these MSI phenotypes in patients.To address this question, we performed tandem affinity purification coupled with mass spectrometry analysis (TAP-MS) to uncover MMR-associated protein complexes. Our proteomics study of the MMR family led to the discovery of many novel MMR-associated proteins, and gene ontology analysis expanded the roles of MMR in multiple biological processes. Specifically for MSH2, we uncovered a novel MutS binding partner WDHD1, which associates with both MutSα (MSH2-MSH6 heterodimer) and MutSβ (MSH2-MSH3 heterodimer). We provide additional evidence suggesting that WDHD1 is involved in the MMR pathway, which can be used as potential biomarker for MSI phenotypes in cancer patients.     

Interactions of Human Mismatch Repair Proteins MutS�� and MutL�� with Proteins of the ATR-Chk1 Pathway

At clinically relevant doses, chemotherapeutic S_N1 DNA methylating agents induce an ATR-mediated checkpoint response in human cells that is dependent on functional MutSα and MutLα. Deficiency of either mismatch repair activity renders cells highly resistant to this class of drug, but the mechanisms linking mismatch repair to checkpoint activation have remained elusive. In this study we have systematically examined the interactions of human MutSα and MutLα with proteins of the ATR-Chk1 pathway using both nuclear extracts and purified proteins. Using nuclear co-immunoprecipitation, we have detected interaction of MutSα with ATR, TopBP1, Claspin, and Chk1 and interaction of MutLα with TopBP1 and Claspin. We were unable to detect interaction of MutSα or MutLα with Rad17, Rad9, or replication protein A in the extract system. Use of purified proteins confirmed direct interaction of MutSα with ATR, TopBP1, and Chk1 and of MutLα with TopBP1. MutSα-Claspin and MutLα-Claspin interactions were not demonstrable with purified proteins, suggesting that extract interactions are indirect or depend on post-translational modification. Use of a modified chromatin immunoprecipitation assay showed that proliferating cell nuclear antigen, ATR, TopBP1, and Chk1 are recruited to chromatin in a MutLα- and MutSα-dependent fashion after N-methyl-N′-nitro-N-nitrosoguanidine treatment. However, chromatin enrichment of replication protein A, Claspin, Rad17-RFC, and Rad9-Rad1-Hus1 was not detected in these experiments. Although our failure to observe enrichment of the latter activities could be due to sensitivity limitations, these observations may indicate a novel mechanism for ATR activation.     

DNA错配修复、染色体不稳定和肿瘤的关系

DNA错配修复系统可以识别并纠正DNA复制过程中出现的错误.保证基因组的稳定性和完整性.错配修复系统缺陷可能导致遗传物质发生突变,引发恶性肿瘤.肿瘤患者经常表现出染色体不稳定,具体表现为微卫星不稳定性和杂合性缺失.本文就DNA错配修复、染色体不稳定和肿瘤之间的联系予以综述.     

DNA错配修复与癌症的发生及治疗

DNA错配修复是细胞复制后的一种修复机制,具有维持DNA复制保真度,控制基因变异的作用。DNA错配修复缺陷使整个基因组不稳定,最终会导致肿瘤和癌症的发生。DNA错配修复系统不仅通过矫正在DNA重组和复制过程中产生的碱基错配而保持基因组的稳定,而且通过诱导DNA损伤细胞的凋亡而消除由突变细胞生长形成的癌变。错配修复缺陷细胞的抗药性也引起了癌症化疗研究方面的关注。大多数情况下,错配修复健全型细胞对肿瘤化疗药物敏感,而错配修复缺陷细胞却有较高的抗性。DNA错配修复系统通过修复和诱导细胞凋亡维护基因组稳定的功能,显示了错配修复途径在癌症生物学和分子医学中的重要性。     

The Amyloid Precursor Protein Controls PIKfyve Function

While the Amyloid Precursor Protein (APP) plays a central role in Alzheimer’s disease, its cellular function still remains largely unclear. It was our goal to establish APP function which will provide insights into APP''s implication in Alzheimer''s disease. Using our recently developed proteo-liposome assay we established the interactome of APP''s intracellular domain (known as AICD), thereby identifying novel APP interactors that provide mechanistic insights into APP function. By combining biochemical, cell biological and genetic approaches we validated the functional significance of one of these novel interactors. Here we show that APP binds the PIKfyve complex, an essential kinase for the synthesis of the endosomal phosphoinositide phosphatidylinositol-3,5-bisphosphate. This signalling lipid plays a crucial role in endosomal homeostasis and receptor sorting. Loss of PIKfyve function by mutation causes profound neurodegeneration in mammals. Using C. elegans genetics we demonstrate that APP functionally cooperates with PIKfyve in vivo. This regulation is required for maintaining endosomal and neuronal function. Our findings establish an unexpected role for APP in the regulation of endosomal phosphoinositide metabolism with dramatic consequences for endosomal biology and important implications for our understanding of Alzheimer''s disease.     

Stochastic Processes and Component Plasticity Governing DNA Mismatch Repair

DNA mismatch repair (MMR) is a DNA excision–resynthesis process that principally enhances replication fidelity. Highly conserved MutS (MSH) and MutL (MLH/PMS) homologs initiate MMR and in higher eukaryotes act as DNA damage sensors that can trigger apoptosis. MSH proteins recognize mismatched nucleotides, whereas the MLH/PMS proteins mediate multiple interactions associated with downstream MMR events including strand discrimination and strand-specific excision that are initiated at a significant distance from the mismatch. Remarkably, the biophysical functions of the MLH/PMS proteins have been elusive for decades. Here we consider recent observations that have helped to define the mechanics of MLH/PMS proteins and their role in choreographing MMR. We highlight the stochastic nature of DNA interactions that have been visualized by single-molecule analysis and the plasticity of protein complexes that employ thermal diffusion to complete the progressions of MMR.     

The Escherichia coli Mismatch Repair Protein MutL Recruits the Vsr and MutH Endonucleases in Response to DNA Damage

The activities of the Vsr and MutH endonucleases of Escherichia coli are stimulated by MutL. The interaction of MutL with each enzyme is enhanced in vivo by 2-aminopurine treatment and by inactivation of the mutY gene. We hypothesize that MutL recruits the endonucleases to sites of DNA damage.The Escherichia coli Dcm protein methylates the second C of CCWGG sites (W = A or T). Deamination of 5-methylcytosine converts CG base pairs to T/G mismatches, causing CCWGG-to-CTWGG transition mutations. Very-short-patch (VSP) repair minimizes these mutations (2). Repair is initiated by a sequence- and mismatch-specific endonuclease, Vsr, which cleaves the DNA 5′ of the T. DNA polymerase I removes the T along with a few 3′ nucleotides and resynthesizes the missing bases, restoring the CG base pair. Vsr is both necessary and sufficient for initiating VSP repair. However, two other proteins, MutS and MutL, enhance VSP repair of deamination damage (1).MutS and MutL are best known for their roles in postreplication mismatch repair (MMR) (9, 11). MutL couples mismatch recognition by MutS to the activation of MutH, an endonuclease that cleaves the unmethylated strand of GATC sequences that are transiently hemimethylated following DNA replication. The nicked strand, containing the erroneous base, is removed by the UvrD helicase and one of several exonucleases to beyond the mismatch and then resynthesized by DNA polymerase III.MutL stimulates the endonuclease activities of both Vsr and MutH in vitro (8, 17). The requirements for stimulation are the same: a mismatch, MutS, and ATP hydrolysis by MutL (8, 8a). Cross-linking studies showed that MutH and Vsr interact with the same region in the N-terminal domain of MutL (Heinze et al., submitted). Competition of Vsr with MutH for access to MutL explains the ability of Vsr to inactivate MMR in vivo when overexpressed (6, 13). Thus, the interactions of the two repair endonucleases with MutL are structurally and functionally very similar.In contrast to MMR, where the cleavage site for MutH may be several kilobases away from the mismatch, VSP repair requires that mismatch recognition and endonucleolytic cleavage occur at the same C(T/G)WGG site. How MutS and MutL stimulate VSP repair if MutS and Vsr compete for the same mismatch remains unknown (2, 12). We hypothesized that MutS binds the mismatch first and that a MutS-MutL complex then recruits Vsr. If so, then the MMR proteins would initially mask the mismatch, making the interaction of Vsr with MutL independent of lesion identity.To test this hypothesis, we studied the interaction of MutL with Vsr and with MutH in response to two types of mismatch by using a bacterial two-hybrid assay (10). This assay detects all known interactions among the Mut proteins: homodimerization of MutS and MutL, interaction of MutL with MutS and with MutH, and interaction of Vsr with the N-terminal domain of MutL (15). We found no false positives or false negatives. Furthermore, since the assay relies on reconstitution of a soluble protein (adenylate cyclase), the DNA repair proteins are free to interact with the DNA (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Known interactions among repair proteins as detected by the bacterial two-hybrid assay. The T18 and T25 subunits of CyaA are fused to any two repair proteins (illustrated here by MutL and Vsr), allowing measurement of all pairwise interactions as units of β-galactosidase (β-gal). T25 fusions are repair proficient. CRP, cyclic AMP (cAMP) receptor protein; P, lac operon promoter; RNAP, RNA polymerase.2-Aminopurine (2AP) mispairs with C during DNA replication, causing transition and frameshift mutations (5). The transitions are due primarily to the mismatch itself; the frameshifts are due to saturation of MMR, which leaves slipped-strand intermediates caused by DNA replication errors unrepaired (19). MutS and MutL bind to 2AP/C lesions (22), although the lesions may not be subject to MMR (19). As shown in Fig. ​Fig.2,2, treatment with 2AP causes a dose-dependent increase in the interaction of MutL with both Vsr and MutH; dimerization of MutL and interaction of MutL with MutS are somewhat increased.Open in a separate windowFIG. 2.Effect of 2AP treatment on protein-protein interactions in the bacterial two-hybrid assay. Results in units of β-galactosidase ± standard errors of the means (n = 9) are shown for BTH101(F galE15 ga1K16 rpsL1 hsdR2 mcrA1 mcrB1 cyaA-99) cells treated with 2AP as described previously (5, 19). Cells were cotransformed with pT18 and pT25 vectors (light gray bars), pT18-mutS and pT25-mutL (white bars), pT18-vsr and pT25-mutL (gray bars), pT18-mutH and pT25-mutL (black bars), or pT18-mutL and pT25-mutL (mottled bars). (NB: The dose-response curve for the pT18-mutS pT25-mutS transformants is similar to that of the pT18-mutL pT25-mutL transformants; it has been omitted for graphical clarity since the MutS-MutS interaction gives very high units of β-galactosidase activity [15]).The MutY adenine glycosylase removes A''s which have mispaired with oxidized guanine (8-oxoG) during DNA replication. Cells with a deletion of mutY have an elevated frequency of CG-to-AT transversion mutations (18); these are reduced by excess MutS, suggesting that 8-oxoG/A mismatches are also subject to MMR (23). As shown in Fig. ​Fig.3,3, the interactions between Vsr and MutL and between MutH and MutL increase in a mutY cell (stippled bars). Other interactions, such as MutS dimerization, are unaffected (not shown).Open in a separate windowFIG. 3.Effects of mutY and mutT deletions on protein-protein interactions in the bacterial two-hybrid assay. Results are in units of β-galactosidase, relative to the level in the wild type, in mutT (solid) and mutY (stippled) derivatives of BTH101 cotransformed with pT18 and pT25 vectors, pT18-mutH and pT25-mutL, pT18-vsr and pT25-mutL, or pT18-mutS and pT25-mutS (n = 3).8-OxoG/A mismatches also arise by incorporation of oxidized dGTP opposite A during DNA replication. The MutT nuclease minimizes this by removing oxidized dGTP from the nucleotide pool. The high frequency of AT-to-CG mutations in mutT strains is unaffected by the status of the MMR system (7, 21, 23), possibly because these 8-oxoG/A mispairs are in a conformation that MutS does not recognize. As shown in Fig. ​Fig.3,3, neither the interaction between MutL and Vsr nor that between MutL and MutH is elevated in a mutT strain (solid bars).These data show that mismatches which attract MutS and MutL increase the interaction of MutL with MutH in vivo. Although these mismatches are not subject to VSP repair, they also increase the interaction between MutL and Vsr. The simplest interpretation is that a MutS-MutL complex recruits MutH and Vsr to the DNA independent of the identity of the mismatch. MutS and MutL could then clear the mismatch, delivering the (activated) endonuclease to its specific target site, no matter how far away it is.Interaction of MutL with MutH, leading to MMR, is probably the default option. However, the MutS-MutL complex may recruit other repair proteins, such as Vsr or UvrB (20), to lesions that are poorly processed by MMR. The T/G mismatch in hemimethylated CTWGG sequences may be one such site. Vsr is expressed at very low levels in growing cells (14), so this recruitment would enhance VSP repair. However, recruitment of Vsr to other lesions would reduce VSP repair. For example, recruitment of Vsr by MutL to 2AP/C lesions (Fig. ​(Fig.2)2) could explain why CCWGG sites are hotspots for 2AP-induced mutations (4, 19).We have argued that Vsr is kept at low levels while DNA is replicating to avoid interference with MMR (14). However, if, as we suggest here, MutS and MutL are needed to recruit scarce Vsr to its target sequence, this argument loses its merit. It seems more likely that Vsr levels are kept low to avoid CTWGG-to-CCWGG mutations; Vsr creates these mutations by converting T/G mismatches formed at CTAGG sites by errors in DNA replication to CG (3, 6, 16). Vsr levels rise in nongrowing cells (14), when mutagenesis is no longer a risk. Under these circumstances, it is likely that MutS and MutL are no longer required for efficient VSP repair.     

Selenium Compounds Activate ATM-dependent DNA Damage Response via the Mismatch Repair Protein hMLH1 in Colorectal Cancer Cells

Epidemiological and animal studies indicate that selenium supplementation suppresses risk of colorectal and other cancers. The majority of colorectal cancers are characterized by a defective DNA mismatch repair (MMR). Here, we have employed the MMR-deficient HCT 116 colorectal cancer cells and the MMR-proficient HCT 116 cells with hMLH1 complementation to investigate the role of hMLH1 in selenium-induced DNA damage response, a tumorigenesis barrier. The ATM (ataxia telangiectasia mutated) protein responds to clastogens and initiates DNA damage response. We show that hMLH1 complementation sensitizes HCT 116 cells to methylseleninic acid, methylselenocysteine, and sodium selenite via reactive oxygen species and facilitates the selenium-induced oxidative 8-oxoguanine damage, DNA breaks, G₂/M checkpoint response, and ATM pathway activation. Pretreatment of the hMLH1-complemented HCT 116 cells with the antioxidant N-acetylcysteine or 2,2,6,6-tetramethylpiperidine-1-oxyl or the ATM kinase inhibitor KU55933 suppresses hMLH1-dependent DNA damage response to selenium exposure. Selenium treatment stimulates the association between hMLH1 and hPMS2 proteins, a heterodimer critical for functional MMR, in a manner dependent on ATM and reactive oxygen species. Taken together, the results suggest a new role of selenium in mitigating tumorigenesis by targeting the MMR pathway, whereby the lack of hMLH1 renders the HCT 116 colorectal cancer cells resistant to selenium-induced DNA damage response.     

Molecular Basis for Phosphorylation-dependent SUMO Recognition by the DNA Repair Protein RAP80

Recognition and repair of double-stranded DNA breaks (DSB) involves the targeted recruitment of BRCA tumor suppressors to damage foci through binding of both ubiquitin (Ub) and the Ub-like modifier SUMO. RAP80 is a component of the BRCA1 A complex, and plays a key role in the recruitment process through the binding of Lys⁶³-linked poly-Ub chains by tandem Ub interacting motifs (UIM). RAP80 also contains a SUMO interacting motif (SIM) just upstream of the tandem UIMs that has been shown to specifically bind the SUMO-2 isoform. The RAP80 tandem UIMs and SIM function collectively for optimal recruitment of BRCA1 to DSBs, although the molecular basis of this process is not well understood. Using NMR spectroscopy, we demonstrate that the RAP80 SIM binds SUMO-2, and that both specificity and affinity are enhanced through phosphorylation of the canonical CK2 site within the SIM. The affinity increase results from an enhancement of electrostatic interactions between the phosphoserines of RAP80 and the SIM recognition module within SUMO-2. The NMR structure of the SUMO-2·phospho-RAP80 complex reveals that the molecular basis for SUMO-2 specificity is due to isoform-specific sequence differences in electrostatic SIM recognition modules.     

Coordinating Multi-Protein Mismatch Repair by Managing Diffusion Mechanics on the DNA

DNA mismatch repair (MMR) corrects DNA base-pairing errors that occur during DNA replication. MMR catalyzes strand-specific DNA degradation and resynthesis by dynamic molecular coordination of sequential downstream pathways. The temporal and mechanistic order of molecular events is essential to insure interactions in MMR that occur over long distances on the DNA. Biophysical real-time studies of highly conserved components on mismatched DNA have shed light on the mechanics of MMR. Single-molecule imaging has visualized stochastically coordinated MMR interactions that are based on thermal fluctuation-driven motions. In this review, we describe the role of diffusivity and stochasticity in MMR beginning with mismatch recognition through strand-specific excision. We conclude with a perspective of the possible research directions that should solve the remaining questions in MMR.