Xiling Wu

hMRE11 deficiency leads to microsatellite instability and defective DNA mismatch repair

DNA mismatch repair (MMR) is essential in the surveillance of accurate transmission of genetic information, and defects in this pathway lead to microsatellite instability and hereditary nonpolyposis colorectal cancer (HNPCC). Our previous study raised the possibility that hMRE11 might be involved in MMR through physical interaction with hMLH1. Here, we show that hMRE11 deficiency leads to significant increase in MSI for both mono‐ and dinucleotide sequences. Furthermore, RNA‐interference‐mediated hMRE11‐knockdown in HeLa cells results in MMR deficiency. Analysis of seven HNPCC‐associated hMLH1 missense mutations located within the hMRE11‐interacting domain shows that four mutations (L574P, K618T, R659P and A681T) cause near‐complete disruption of the interaction between hMRE11 and hMLH1, and two mutations (Q542L and L582V) cause a 30% reduction of protein interaction. These findings indicate that hMRE11 represents a functional component of the MMR pathway and the disruption of hMLH1–hMRE11 interaction could be an alternative molecular explanation for hMLH1 mutations in a subset of HNPCC tumours.

MutS homologues hMSH4 and hMSH5: diverse functional implications in humans.

The DNA mismatch repair (MMR) pathway is one of the most critical genome surveillance systems for governing faithful transmission of genetic information during DNA replication. The functional necessity of this pathway in humans is partially reflected by the tight link between MMR gene mutations and the development of hereditary nonpolyposis colorectal cancer. Increasing evidence has suggested a broad involvement of MMR proteins in various aspects of DNA metabolism beyond the scope of DNA mismatch correction, such as in the processes of DNA damage response and homologous recombination. Though evidence is presently lacking for potential functional involvement of hMSH4 and hMSH5 in MMR, these two proteins are thought to play roles in meiotic and mitotic DNA double strand break (DSB) repair and DNA damage responses in human cells.

Evidence for a direct involvement of hMSH5 in promoting ionizing radiation induced apoptosis

Although increasing evidence has suggested that the hMSH5 protein plays an important role in meiotic and mitotic DNA recombinational repair, its precise functions in recombination and DNA damage response are presently elusive. Here we show that the interaction between hMSH5 and c-Abl confers ionizing radiation (IR)-induced apoptotic response by promoting c-Abl activation and p73 accumulation, and these effects are greatly enhanced in cells expressing hMSH5(P29S) (i.e. the hMSH5 variant possessing a proline to serine change within the N-terminal (Px)(5) dipeptide repeat). Our current study provides the first evidence that the (Px)(5) dipeptide repeat plays an important role in modulating the interaction between hMSH5 and c-Abl and alteration of this dipeptide repeat in hMSH5(P29S) leads to increased IR sensitivity owing to enhanced caspase-3-mediated apoptosis. In addition, RNAi-mediated hMSH5 silencing leads to the reduction of apoptosis in IR-treated cells. In short, this study implicates a role for hMSH5 in DNA damage response involving c-Abl and p73, and suggests that mutations impairing this process could significantly affect normal cellular responses to anti-cancer treatments.

Physical and Functional Interaction between hMSH5 and c-Abl

Despite being a member of the mismatch repair family of proteins, the biological functions of hMSH5 in human cells are presently elusive. Here, we report a novel physical and functional interaction between hMSH5 and c-Abl; the latter is a critical non-receptor tyrosine kinase involved in many critical cellular functions including DNA damage response, in which the kinase activity is normally suppressed in the absence of biological challenges. Our data indicate that hMSH5 associates with c-Abl in vivo, which is mediated by a direct physical interaction between the NH2 terminus (residues 1-109) of hMSH5 and the c-Abl SH3 domain. This physical interaction facilitates the activation of c-Abl tyrosine kinase and the phosphorylation of hMSH5 in response to ionizing radiation. Our data also indicate that the hMSH5 P29S variant overactivates the c-Abl tyrosine kinase activity. Furthermore, it seems that the tyrosine phosphorylation of hMSH5 promotes the dissociation of hMSH4-hMSH5 heterocomplex. Together, the revealed physical and functional interaction of hMSH5 with c-Abl implies that the interplay between hMSH5 and c-Abl could manipulate cellular responses to ionizing radiation-induced DNA damages.

MutS Homologues hMSH4 and hMSH5: Genetic Variations, Functions, and Implications in Human Diseases

The prominence of the human mismatch repair (MMR) pathway is clearly reflected by the causal link between MMR gene mutations and the occurrence of Lynch syndrome (or HNPCC). The MMR family of proteins also carries out a plethora of diverse cellular functions beyond its primary role in MMR and homologous recombination. In fact, members of the MMR family of proteins are being increasingly recognized as critical mediators between DNA damage repair and cell survival. Thus, a better functional understanding of MMR proteins will undoubtedly aid the development of strategies to effectively enhance apoptotic signaling in response to DNA damage induced by anti-cancer therapeutics. Among the five known human MutS homologs, hMSH4 and hMSH5 form a unique heterocomplex. However, the expression profiles of the two genes are not correlated in a number of cell types, suggesting that they may function independently as well. Consistent with this, these two proteins are promiscuous and thought to play distinct roles through interacting with different binding partners. Here, we describe the gene and protein structures of eukaryotic MSH4 and MSH5 with a particular emphasis on their human homologues, and we discuss recent findings of the roles of these two genes in DNA damage response and repair. Finally, we delineate the potential links of single nucleotide polymorphism (SNP) loci of these two genes with several human diseases.

MutS homologue hMSH5: role in cisplatin-induced DNA damage response

BackgroundCisplatin (cis-diamminedichloroplatinum (II), CDDP) and its analogues constitute an important class of anticancer drugs in the treatment of various malignancies; however, its effectiveness is frequently affected by mutations in genes involved in the repair and signaling of cisplatin-induced DNA damage. These observations necessitate a need for a better understanding of the molecular events governing cellular sensitivity to cisplatin.ResultsHere, we show that hMSH5 mediates sensitization to cisplatin-induced DNA damage in human cells. Our study indicates that hMSH5 undergoes cisplatin-elicited protein induction and tyrosine phosphorylation. Silencing of hMSH5 by RNAi or expression of hMSH5 phosphorylation-resistant mutant hMSH5Y742F elevates cisplatin-induced G2 arrest and renders cells susceptible to cisplatin toxicity at clinically relevant doses. In addition, our data show that cisplatin promotes hMSH5 chromatin association and hMSH5 deficiency increases cisplatin-triggered γ-H2AX foci. Consistent with a possible role for hMSH5 in recombinational repair of cisplatin-triggered double-strand breaks (DSBs), the formation of cisplatin-induced hMSH5 nuclear foci is hRad51-dependent.ConclusionCollectively, our current study has suggested a role for hMSH5 in the processing of cisplatin-induced DSBs, and silencing of hMSH5 may provide a new means to improve the therapeutic efficacy of cisplatin.

MutS homologue hMSH4: interaction with eIF3f and a role in NHEJ-mediated DSB repair

BackgroundDNA mismatch repair proteins participate in diverse cellular functions including DNA damage response and repair. As a member of this protein family, the molecular mechanisms of hMSH4 in mitotic cells are poorly defined. It is known that hMSH4 is promiscuous, and among various interactions the hMSH4-hMSH5 interaction is involved in recognizing DNA intermediate structures arising from homologous recombination (HR).ResultsWe identified a new hMSH4 interacting protein eIF3f – a protein that functions not only in translation but also in the regulation of apoptosis and tumorigenesis in humans. Our studies have demonstrated that hMSH4-eIF3f interaction is mediated through the N-terminal regions of both proteins. The interaction with eIF3f fosters hMSH4 protein stabilization, which in turn sustains γ-H2AX foci and compromises cell survival in response to ionizing radiation (IR)-induced DNA damage. These effects can be, at least partially, attributed to the down-regulation of NHEJ activity by hMSH4. Furthermore, the interplay between hMSH4 and eIF3f inhibits IR-induced AKT activation, and hMSH4 promotes eIF3f-mediated bypass of S phase arrest, and ultimately enhancing an early G2/M arrest in response to IR treatment.ConclusionOur current study has revealed a role for hMSH4 in the maintenance of genomic stability by suppressing NHEJ-mediated DSB repair.

Assessment of Anti-recombination and Double-strand Break-induced Gene Conversion in Human Cells by a Chromosomal Reporter

Background: DSB repair is frequently associated with gene conversion. Results: Gene conversions at the site of a DSB and its surrounding regions are regulated differently. Conclusion: hMSH2, hMLH1, and hMRE11 play different roles in proximal and distal gene conversions. Significance: Delineating the mechanisms underlying DSB-induced gene conversion will help to decipher how mutations in DSB repair genes affect genome stability. Gene conversion is one of the frequent end results of homologous recombination, and it often underlies the inactivation of tumor suppressor genes in cancer cells. Here, we have developed an integrated assay system that allows simultaneous examination of double-strand break (DSB)-induced gene conversion events at the site of a DSB (proximal region) and at a surrounding region ∼1 kb away from the break (distal region). Utilizing this assay system, we find that gene conversion events at the proximal and distal regions are relatively independent of one another. The results also indicate that synthesis-dependent strand annealing (SDSA) plays a major role in DSB-induced gene conversion. In addition, our current study has demonstrated that hMLH1 plays an essential role in anti-recombination and gene conversion. Specifically, the anti-recombination activity of hMLH1 is partially dependent on its interaction with hMRE11. Our data suggests that the role of hMLH1 and hMRE11 in the process of gene conversion is complex, and these proteins play different roles in DSB-induced proximal and distal gene conversions. In particular, the involvement of hMLH1 and hMRE11 in the distal gene conversion requires both hMSH2 and heteroduplex formation.

hMSH5 Facilitates the Repair of Camptothecin-induced Double-strand Breaks through an Interaction with FANCJ.

Background: Camptothecin induces replication-associated DSB formation. Results: hMSH5-FANCJ facilitates the repair of camptothecin-induced DSBs. Conclusion: Functional interplay between hMSH5 and FANCJ is involved in replication stress-induced DSB repair. Significance: Understanding the mechanisms of DSB repair, induced by replication stress, is pivotal to develop new anticancer targets and therapeutic strategies. Replication stress from stalled or collapsed replication forks is a major challenge to genomic integrity. The anticancer agent camptothecin (CPT) is a DNA topoisomerase I inhibitor that causes fork collapse and double-strand breaks amid DNA replication. Here we report that hMSH5 promotes cell survival in response to CPT-induced DNA damage. Cells deficient in hMSH5 show elevated CPT-induced γ-H2AX and RPA2 foci with concomitant reduction of Rad51 foci, indicative of impaired homologous recombination. In addition, CPT-treated hMSH5-deficient cells exhibit aberrant activation of Chk1 and Chk2 kinases and therefore abnormal cell cycle progression. Furthermore, the hMSH5-FANCJ chromatin recruitment underlies the effects of hMSH5 on homologous recombination and Chk1 activation. Intriguingly, FANCJ depletion desensitizes hMSH5-deficient cells to CPT-elicited cell killing. Collectively, our data point to the existence of a functional interplay between hMSH5 and FANCJ in double-strand break repair induced by replication stress.

MutS Homologue hMSH5: Recombinational DSB Repair and Non-Synonymous Polymorphic Variants

Double-strand breaks (DSBs) constitute the most deleterious form of DNA lesions that can lead to genome alterations and cell death, and the vast majority of DSBs arise pathologically in response to DNA damaging agents such as ionizing radiation (IR) and chemotherapeutic agents. Recent studies have implicated a role for the human MutS homologue hMSH5 in homologous recombination (HR)-mediated DSB repair and the DNA damage response. In the present study, we show that hMSH5 promotes HR-based DSB repair, and this property resides in the carboxyl-terminal portion of the protein. Our results demonstrate that DSB-triggered hMSH5 chromatin association peaks at the proximal regions of the DSB and decreases gradually with increased distance from the break. Furthermore, the DSB-triggered hMSH5 chromatin association is preceded by and relies on the assembly of hMRE11 and hRad51 at the proximal regions of the DSB. Lastly, the potential effects of hMSH5 non-synonymous variants (L85F, Y202C, V206F, R351G, L377F, and P786S) on HR and cell survival in response to DSB-inducing anticancer agents have been analyzed. These experiments show that the expression of hMSH5 variants elicits different survival responses to anticancer drugs cisplatin, bleomycin, doxorubicin and camptothecin. However, the effects of hMSH5 variants on survival responses to DSB-inducing agents are not directly correlated to their effects exerted on HR-mediated DSB repair, suggesting that the roles of hMSH5 variants in the processes of DNA damage response and repair are multifaceted.