[Show abstract][Hide abstract] ABSTRACT: MutS protein homolog 2 (MSH2) is a key DNA mismatch repair protein. It forms the MSH2-MSH6 (MutSα) and MSH2-MSH3 (MutSβ) heterodimers, which help to ensure genomic integrity. MutSα not only recognizes and repairs mismatched nucleotides but also recognizes DNA adducts induced by DNA-damaging agents, and triggers cell-cycle arrest and apoptosis. Loss or depletion of MutSα from cells leads to microsatellite instability (MSI) and resistance to DNA damage. Although the level of MutSα can be reduced by the ubiquitin-proteasome pathway, the detailed mechanisms of this regulation remain elusive. Here we report that histone deacetylase 6 (HDAC6) sequentially deacetylates and ubiquitinates MSH2, leading to MSH2 degradation. In addition, HDAC6 significantly reduces cellular sensitivity to DNA-damaging agents and decreases cellular DNA mismatch repair activities by downregulation of MSH2. Overall, these findings reveal a mechanism by which proper levels of MutSα are maintained.
[Show abstract][Hide abstract] ABSTRACT: DNA mismatch repair (MMR) ensures replication fidelity by correcting mismatches generated during DNA replication. Although human MMR has been reconstituted in vitro, how MMR occurs in vivo is unknown. Here, we show that an epigenetic histone mark, H3K36me3, is required in vivo to recruit the mismatch recognition protein hMutSα (hMSH2-hMSH6) onto chromatin through direct interactions with the hMSH6 PWWP domain. The abundance of H3K36me3 in G1 and early S phases ensures that hMutSα is enriched on chromatin before mispairs are introduced during DNA replication. Cells lacking the H3K36 trimethyltransferase SETD2 display microsatellite instability (MSI) and an elevated spontaneous mutation frequency, characteristic of MMR-deficient cells. This work reveals that a histone mark regulates MMR in human cells and explains the long-standing puzzle of MSI-positive cancer cells that lack detectable mutations in known MMR genes.
[Show abstract][Hide abstract] ABSTRACT: Expansion of CAG/CTG trinucleotide repeats causes certain familial neurological disorders. Hairpin formation in the nascent strand during DNA synthesis is considered a major path for CAG/CTG repeat expansion. However, the underlying mechanism is unclear. We show here that removal or retention of a nascent strand hairpin during DNA synthesis depends on hairpin structures and types of DNA polymerases. Polymerase (pol) δ alone removes the 3'-slipped hairpin using its 3'-5' proofreading activity when the hairpin contains no immediate 3' complementary sequences. However, in the presence of pol β, pol δ preferentially facilitates hairpin retention regardless of hairpin structures. In this reaction, pol β incorporates several nucleotides to the hairpin 3' end, which serves as an effective primer for the continuous DNA synthesis by pol δ, thereby leading to hairpin retention and repeat expansion. These findings strongly suggest that coordinated processing of 3'-slipped (CAG)n/(CTG)n hairpins by polymerases δ and β on during DNA synthesis induces CAG/CTG repeat expansions.
Journal of Biological Chemistry 04/2013; · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Expansion of CAG/CTG repeats causes certain neurological and neurodegenerative disorders, and the formation and subsequent persistence of stable DNA hairpins within these repeats are believed to contribute to CAG/CTG repeat instability. Human cells possess a DNA hairpin repair (HPR) pathway, which removes various (CAG)(n) and (CTG)(n) hairpins in a nick-directed and strand-specific manner. Interestingly, this HPR system processes a (CTG)(n) hairpin on the template DNA strand much less efficiently than a (CAG)(n) hairpin on the same strand (Hou, C., Chan, N. L., Gu, L., and Li, G. M. (2009) Incision-dependent and error-free repair of (CAG)(n)/(CTG)(n) hairpins in human cell extracts. Nat. Struct. Mol. Biol. 16, 869-875), suggesting the involvement of an additional component for (CTG)(n) HPR. To identify this activity, a functional in vitro HPR assay was used to screen partially purified HeLa nuclear fractions for their ability to stimulate (CTG)(n) HPR. We demonstrate here that the stimulating activity is the Werner syndrome protein (WRN). Although WRN contains both a 3'→5' helicase activity and a 3'→5' exonuclease activity, the stimulating activity was found to be the helicase activity, as a WRN helicase mutant failed to enhance (CTG)(n) HPR. Consistently, WRN efficiently unwound large (CTG)(n) hairpins and promoted DNA polymerase δ-catalyzed DNA synthesis using a (CTG)(n) hairpin as a template. We, therefore, conclude that WRN stimulates (CTG)(n) HPR on the template DNA strand by resolving the hairpin so that it can be efficiently used as a template for repair or replicative synthesis.
Journal of Biological Chemistry 07/2012; 287(36):30151-6. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The hMSH2(M688R) mismatch repair (MMR) gene mutation has been found in five large families from Tenerife, Spain, suggesting it is a Lynch syndrome or hereditary non-polyposis colorectal cancer (LS/HNPCC) founder mutation. In addition to classical LS/HNPCC tumors, these families present with a high incidence of central nervous system (CNS) tumors normally associated with Turcot or constitutional mismatch repair deficiency (CMMR-D) syndromes. Turcot and CMMR-D mutations may be biallelic, knocking out both copies of the MMR gene. The hMSH2(M688R) mutation is located in the ATP hydrolysis (ATPase) domain. We show that the hMSH2(M688R)-hMSH6 heterodimer binds to mismatched nucleotides but lacks normal ATP functions and inhibits MMR in vitro when mixed with the wild-type (WT) heterodimer. Another alteration that has been associated with LS/HNPCC, hMSH2(M688I)-hMSH6, displays no identifiable differences with the WT heterodimer. Interestingly, some extracolonic tumors from hMSH2(M688R) carriers may express hMSH2-hMSH6, yet display microsatellite instability (MSI). The functional analysis along with variability in tumor expression and the high incidence of CNS tumors suggests that hMSH2(M688R) may act as a dominant negative in some tissues, while the hMSH2(M688I) is most likely a benign polymorphism.
[Show abstract][Hide abstract] ABSTRACT: Expansion of CAG/CTG trinucleotide repeats (TNRs) in humans is associated with a number of neurological and neurodegenerative disorders including Huntington's disease. Increasing evidence suggests that formation of a stable DNA hairpin within CAG/CTG repeats during DNA metabolism leads to TNR instability. However, the molecular mechanism by which cells recognize and repair CAG/CTG hairpins is largely unknown. Recent studies have identified a novel DNA repair pathway specifically removing (CAG)(n)/(CTG)(n) hairpins, which is considered a major mechanism responsible for TNR instability. The hairpin repair (HPR) system targets the repeat tracts for incisions in the nicked strand in an error-free manner. To determine the substrate spectrum of the HPR system and its ability to process smaller hairpins, which may be the intermediates for CAG/CTG expansions, we constructed a series of CAG/CTG hairpin heteroduplexes containing different numbers of repeats (from 5 to 25) and examined their repair in human nuclear extracts. We show here that although repair efficiencies differ slightly among these substrates, removal of the individual hairpin structures all involve endonucleolytic incisions within the repeat tracts in the nicked DNA strand. Analysis of the repair intermediates defined specific incision sites for each substrate, which were all located within the repeat regions. Mismatch repair proteins are not required for, nor do they inhibit, the processing of smaller hairpin structures. These results suggest that the HPR system ensures CAG/CTG stability primarily by removing various sizes of (CAG)(n)/(CTG)(n) hairpin structures during DNA metabolism.
DNA repair 02/2012; 11(2):201-9. · 4.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) are critical post-transcriptional regulators and are derived from hairpin-shaped primary transcripts via a series of processing steps. However, how the production of individual miRNAs is regulated remains largely unknown. Similarly, loss or overexpression of the key mismatch repair protein MutLα (MLH1-PMS2 heterodimer) leads to genome instability and tumorigenesis, but the mechanisms controlling MutLα expression are unknown. Here we demonstrate in vitro and in vivo that MLH1 and miR-422a participate in a feedback loop that regulates the level of both molecules. Using a defined in-vitro miRNA processing system, we show that MutLα stimulates the conversion of pri-miR-422a to pre-miR-422a, as well as the processing of other miRNAs tested, implicating MutLα as a general stimulating factor for miRNA biogenesis. This newly identified MutLα function requires its ATPase and pri-miRNA binding activities. In contrast, miR-422a downregulates MutLα levels by suppressing MLH1 expression through base pairing with the MLH1 3'-untranslated region. A model depicting this feedback mechanism is discussed.
Cell Research 01/2012; 22(6):973-85. · 10.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Acrolein (Acr), a ubiquitous environmental contaminant, is a human carcinogen. Acr can react with DNA to form mutagenic α- and γ-hydroxy-1, N(2)-cyclic propano-2'-deoxyguanosine adducts (α-OH-Acr-dG and γ-OH-Acr-dG). We demonstrate here that Acr-dG adducts can be efficiently repaired by the nucleotide excision repair (NER) pathway in normal human bronchial epithelia (NHBE) and lung fibroblasts (NHLF). However, the same adducts were poorly processed in cell lysates isolated from Acr-treated NHBE and NHLF, suggesting that Acr inhibits NER. In addition, we show that Acr treatment also inhibits base excision repair and mismatch repair. Although Acr does not change the expression of XPA, XPC, hOGG1, PMS2 or MLH1 genes, it causes a reduction of XPA, XPC, hOGG1, PMS2, and MLH1 proteins; this effect, however, can be neutralized by the proteasome inhibitor MG132. Acr treatment further enhances both bulky and oxidative DNA damage-induced mutagenesis. These results indicate that Acr not only damages DNA but can also modify DNA repair proteins and further causes degradation of these modified repair proteins. We propose that these two detrimental effects contribute to Acr mutagenicity and carcinogenicity.
Journal of Biological Chemistry 01/2012; 287(15):12379-86. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mismatch repair corrects biosynthetic errors generated during DNA replication. Mismatch repair deficiency causes a mutator phenotype and directly underlies hereditary nonpolyposis colorectal cancer and some sporadic cancers. Because of remarkably high conservation of the mismatch repair machinery between the budding yeast (Saccharomyces cerevisiae) and humans, the study of mismatch repair in yeast has provided tremendous insights into the mechanisms of this repair pathway in humans. Here we describe a set of practical protocols for how to prepare the yeast and HeLa cell-free nuclear extracts and site-specific DNA mismatch substrates, and how to carry out the in vitro mismatch repair assay. We validated the yeast cell-free system by the mismatch repair deficient strain (Δmsh2) and the complementation assay with purified yeast MutSα.
[Show abstract][Hide abstract] ABSTRACT: The in vitro DNA mismatch repair (MMR) assay is a very useful technique for studying the functions and the mechanisms of the MMR system in genome maintenance. This assay has been effectively used to evaluate MMR proficiency in various tumor cells and to identify the majority of the protein components required for MMR. The procedure for setting up and performing the MMR assay involves mismatch substrate preparation, cell extract preparation, and the repair assay. In this chapter, we describe the detailed methods for this functional in vitro assay.
[Show abstract][Hide abstract] ABSTRACT: During DNA replication or repair, disease-associated (CAG)n/(CTG)n expansion can result from formation of hairpin structures in the repeat tract of the newly synthesized or nicked DNA strand. Recent studies identified a nick-directed (CAG)n/(CTG)n hairpin repair (HPR) system that removes (CAG)n/(CTG)n hairpins from human cells via endonucleolytic incisions. Because the process is highly similar to the mechanism by which XPG and XPF endonucleases remove bulky DNA lesions during nucleotide excision repair, we assessed the potential role of XPG in conducting (CAG)n/(CTG)n HPR.
To determine if the XPG endonuclease is involved in (CAG)n/(CTG)n hairpin removal, two XPG-deficient cell lines (GM16024 and AG08802) were examined for their ability to process (CAG)n/(CTG)n hairpins in vitro. We demonstrated that the GM16024 cell line processes all hairpin substrates as efficiently as HeLa cells, and that the AG08802 cell line is partially defective in HPR. Analysis of repair intermediates revealed that nuclear extracts from both XPG-deficient lines remove CAG/CTG hairpins via incisions, but the incision products are distinct from those generated in HeLa extracts. We also show that purified recombinant XPG protein greatly stimulates HPR in XPG-deficient extracts by promoting an incision 5' to the hairpin.
Our results strongly suggest that 1) human cells possess multiple pathways to remove (CAG)n/(CTG)n hairpins located in newly synthesized (or nicked) DNA strand; and 2) XPG, although not essential for (CAG)n/(CTG)n hairpin removal, stimulates HPR by facilitating a 5' incision to the hairpin. This study reveals a novel role for XPG in genome-maintenance and implicates XPG in diseases caused by trinucleotide repeat expansion.
[Show abstract][Hide abstract] ABSTRACT: The influence of chromatin structure on DNA metabolic processes, including DNA replication and repair, has been a matter of intensive studies in recent years. Although the human mismatch repair (MMR) reaction has been reconstituted using purified proteins, the influence of chromatin structure on human MMR is unknown. This study examines the interaction between human MutSalpha and a mismatch located within a nucleosome or between two nucleosomes. The results show that, whereas MutSalpha specifically recognizes both types of nucleosomal heteroduplexes, the protein bound the mismatch within a nucleosome with much lower efficiency than a naked heteroduplex or a heterology free of histone proteins but between two nucleosomes. Additionally, MutSalpha displays reduced ATPase- and ADP-binding activity when interacting with nucleosomal heteroduplexes. Interestingly, nucleosomes block ATP-induced MutSalpha sliding along the DNA helix when the mismatch is in between two nucleosomes. These findings suggest that nucleosomes may inhibit MMR in eukaryotic cells. The implications of these findings for our understanding of eukaryotic MMR are discussed.
Journal of Biological Chemistry 10/2009; 284(48):33056-61. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Expansion of CAG/CTG trinucleotide repeats is associated with certain familial neurological disorders, including Huntington's disease. Increasing evidence suggests that formation of a stable DNA hairpin within CAG/CTG repeats during DNA metabolism contributes to their expansion. However, the molecular mechanism(s) by which cells remove CAG/CTG hairpins remain unknown. Here we demonstrate that human cell extracts can catalyze error-free repair of CAG/CTG hairpins in a nick-directed manner. The repair system specifically targets CAG/CTG tracts for incisions in the nicked DNA strand, followed by DNA resynthesis using the continuous strand as a template, thereby ensuring CAG/CTG stability. Proliferating cell nuclear antigen (PCNA) is required for the incision step of the hairpin removal, which uses distinct endonuclease activities for individual CAG/CTG hairpins depending on their strand locations and/or secondary structures. We discuss the implications of these data for understanding the etiology of neurological diseases and trinucleotide repeat instability.
[Show abstract][Hide abstract] ABSTRACT: CAG repeats form stable hairpin structures, which are believed to be responsible for CAG repeat expansions associated with certain human neurological diseases. Human cells possess an accurate DNA hairpin repair system that prevents expansion of disease-associated CAG repeats. Based on transgenic animal studies, it is suggested that (CAG)(n) expansion is caused by abnormal binding of the MutSbeta mismatch recognition protein to (CAG)(n) hairpins, leading to hijacking mismatch repair function during (CAG)(n) hairpin repair. We demonstrate here that MutSbeta displays identical biochemical and biophysical activities (including ATP-provoked conformational change, ATPase, ATP binding, and ADP binding) when interacting with a (CAG)(n) hairpin and a mismatch. More importantly, our in vitro functional hairpin repair assays reveal that excess MutSbeta does not inhibit (CAG)(n) hairpin repair in HeLa nuclear extracts. Evidence presented here provides a novel view as to whether or not MutSbeta is involved in CAG repeat instability in humans.
Journal of Biological Chemistry 07/2009; 284(31):20452-6. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: MutSalpha (MSH2/MSH6) and MutSbeta (MSH2/MSH3) are eukaryotic mismatch recognition proteins that preferentially process base-base and small insertion/deletion (ID) mispairs, respectively, despite the fact that cells contain a MutSalpha:MutSbeta ratio of 10:1. To explore the mechanism underlying the differential mismatch recognition by these two proteins, purified human MutSalpha and MutSbeta were analyzed individually and competitively for their abilities to interact with a T-G and an ID substrate. We show that MutSalpha has K(D) values of 26.5 and 38.2 nm for the G-T and ID substrates, respectively, and that MutSbeta has K(D) values of 76.5 and 23.5 nm for G-T and ID, respectively. Consistent with these results, competitive binding assays revealed the following relative binding affinities: MutSbeta-ID > MutSalpha-T-G > MutSalpha-ID > MutSbeta-T-G. Interestingly, binding of MutSbeta to ID heteroduplexes is greatly stimulated when the MutSalpha:MutSbeta ratio is > or = 10. Distinct ATP/ADP binding and ATPase activities of MutSalpha and MutSbeta were also observed. In the absence of DNA, ADP binding and ATPase activities of MutSbeta are significantly higher than those of MutSalpha. However, interaction with DNA significantly stimulates the MutSalpha ATPase activity and reduces the MutSbeta ATPase activity, the consequence being that both proteins exhibit the same level of hydrolytic activity. We conclude that the preferential processing of base-base and ID heteroduplexes by MutSalpha and MutSbeta is determined by their significant differences in ATPase activity, ADP binding activity, and high cellular MutSalpha:MutSbeta ratio.
Journal of Biological Chemistry 03/2009; 284(17):11557-62. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mismatch repair corrects biosynthetic errors generated during DNA replication, whose deficiency causes a mutator phenotype and directly underlies hereditary non-polyposis colorectal cancer and sporadic cancers. Because of remarkably high conservation of the mismatch repair machinery between the budding yeast (Saccharomyces cerevisiae) and humans, the study of mismatch repair in yeast has provided tremendous insights into the mechanisms of this repair pathway in humans. In addition, yeast cells possess an unbeatable advantage over human cells in terms of the easy genetic manipulation, the availability of whole genome deletion strains, and the relatively low cost for setting up the system. Although many components of eukaryotic mismatch repair have been identified, it remains unclear if additional factors, such as DNA helicase(s) and redundant nuclease(s) besides EXO1, participate in eukaryotic mismatch repair. To facilitate the discovery of novel mismatch repair factors, we developed a straightforward in vitro cell-free repair system. Here, we describe the practical protocols for preparation of yeast cell-free nuclear extracts and DNA mismatch substrates, and the in vitro mismatch repair assay. The validity of the cell-free system was confirmed by the mismatch repair deficient yeast strain (Deltamsh2) and the complementation assay with purified yeast MSH2-MSH6.
[Show abstract][Hide abstract] ABSTRACT: Eight-hydroxy-2'-deoxyguanosine (8-OHdG) is increased in the brain in late-stage Alzheimer's disease (LAD) and mild cognitive impairment (MCI). To determine if decreased base-excision repair contributes to these elevations, we measured oxoguanine glycosylase 1 (OGG1) protein and incision activities in nuclear and mitochondrial fractions from frontal (FL), temporal (TL), and parietal (PL) lobes from 8 MCI and 7 LAD patients, and 6 age-matched normal control (NC) subjects. OGG1 activity was significantly (P<0.05) decreased in nuclear specimens of FL, TL, and PL in MCI and LAD and in mitochondria from LAD FL and TL and MCI TL. Nuclear OGG1 protein was significantly decreased in LAD FL and MCI and LAD PL. No differences in mitochondrial OGG1 protein levels were found. Overall, our results suggest that decreased OGG1 activity occurs early in the progression of AD, possibly mediated by 4-hydroxynonenal inactivation and may contribute to elevated 8-OHdG in the brain in MCI and LAD.
Free Radical Biology and Medicine 06/2008; 45(6):813-9. · 5.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This report describes the identification and purification of a novel mismatch repair stimulatory factor from HeLa cell extracts. This activity copurifies with a proliferating cell nuclear antigen-dependent 5 ' --> 3 ' DNA excision activity during several purification steps but is resolved from the excision activity during gel filtration chromatography using Sephacryl S-300. After purification to near homogeneity, the stimulatory factor is associated with three polypeptides with apparent molecular masses of 68, 36, and 30 kDa. Peptide sequencing analysis by tandem mass spectrometry identified the stimulatory factor as the heterotrimeric regulatory factor X (RFX) complex, which regulates transcription of the class II major histocompatibility complex by facilitating histone acetylation and is defective in the human hereditary immunodeficiency syndrome called bare lymphocyte syndrome. This conclusion was confirmed by the facts that purified recombinant RFX stimulates mismatch repair in an in vitro reconstituted mismatch repair system and that depletion of RFX from nuclear extracts or RFX knockdown in cells reduces mismatch repair activity. As expected, RFX knockdown cells display instability in microsatellite sequences. The possible role of RFX in human MMR repair is discussed.
Journal of Biological Chemistry 06/2008; 283(19):12730-5. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Our previous studies indicate that hMRE11 plays a role in MMR, and this function of hMRE11 is most likely mediated by the hMLH1-hMRE11 interaction. Here, we explored the functional implications of the hMLH1-hMRE11 interaction in MMR and the effects of hMLH1 mutations on their interaction. Our in vitro MMR assay demonstrated that the dominant-negative hMRE11(452-634) mutant peptide (i.e., harboring only the hMLH1-interacting domain) imparted a significant reduction in both 3' excision and 3'-directed MMR activities. Furthermore, the expression of hMRE11(452-634), and to a lesser extent hMRE11(1-634) (ATLD1), impaired G2/M checkpoint control in response to MNU and cisplatin treatments, rendering cells resistant to killings by these two anticancer drugs. Analysis of 38 hMLH1 missense mutations showed that the majority of mutations caused significant (>50%) reductions in their interaction with hMRE11, suggesting a potential link between aberrant protein interaction and the pathogenic effects of hMLH1 variants.
Biochemical and Biophysical Research Communications 06/2008; 370(2):338-43. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Acute myeloid leukemia (AML) is an aggressive hematological cancer. Despite therapeutic regimens that lead to complete remission, the vast majority of patients undergo relapse. The molecular mechanisms underlying AML development and relapse remain incompletely defined. To explore whether loss of DNA mismatch repair (MMR) function is involved in AML, we screened two key MMR genes, MSH2 and MLH1, for mutations and promoter hypermethylation in leukemia specimens from 53 AML patients and blood from 17 non-cancer controls. We show here that whereas no amino acid alteration or promoter hypermethylation was detected in all control samples, 18 AML patients exhibited either mutations in MMR genes or hypermethylation in the MLH1 promoter. In vitro functional MMR analysis revealed that almost all the mutations analyzed resulted in loss of MMR function. MMR defects were significantly more frequent in patients with refractory or relapsed AML compared with newly diagnosed patients. These observations suggest for the first time that the loss of MMR function is associated with refractory and relapsed AML and may contribute to disease pathogenesis.
Cell Research 03/2008; 18(2):281-9. · 10.53 Impact Factor