Peggy Hsieh

DNA mismatch repair: molecular mechanism, cancer, and ageing.

DNA mismatch repair (MMR) proteins are ubiquitous players in a diverse array of important cellular functions. In its role in post-replication repair, MMR safeguards the genome correcting base mispairs arising as a result of replication errors. Loss of MMR results in greatly increased rates of spontaneous mutation in organisms ranging from bacteria to humans. Mutations in MMR genes cause hereditary nonpolyposis colorectal cancer, and loss of MMR is associated with a significant fraction of sporadic cancers. Given its prominence in mutation avoidance and its ability to target a range of DNA lesions, MMR has been under investigation in studies of ageing mechanisms. This review summarizes what is known about the molecular details of the MMR pathway and the role of MMR proteins in cancer susceptibility and ageing.

Composite Active Site of an ABC ATPase: MutS Uses ATP to Verify Mismatch Recognition and Authorize DNA Repair

The MutS protein initiates DNA mismatch repair by recognizing mispaired and unpaired bases embedded in duplex DNA and activating endo- and exonucleases to remove the mismatch. Members of the MutS family also possess a conserved ATPase activity that belongs to the ATP binding cassette (ABC) superfamily. Here we report the crystal structure of a ternary complex of MutS-DNA-ADP and assays of initiation of mismatch repair in conjunction with perturbation of the composite ATPase active site by mutagenesis. These studies indicate that MutS has to bind both ATP and the mismatch DNA simultaneously in order to activate the other mismatch repair proteins. We propose that the MutS ATPase activity plays a proofreading role in DNA mismatch repair, verification of mismatch recognition, and authorization of repair.

Molecular mechanisms of DNA mismatch repair

DNA mismatch repair (MMR) safeguards the integrity of the genome. In its role in postreplicative repair, this repair pathway corrects base-base and insertion/deletion (I/D) mismatches that have escaped the proofreading function of replicative polymerases. In its absence, cells assume a mutator phenotype in which the rate of spontaneous mutation is greatly elevated. The discovery that defects in mismatch repair segregate with certain cancer predisposition syndromes highlights its essential role in mutation avoidance. Recently, three-dimensional structures of MutS, a key repair protein that recognizes mismatches, have been determined by X-ray crystallography. This article provides an overview of the structural features of MutS proteins and discusses how the structural data together with biochemical and genetic studies reveal new insights into the molecular mechanisms of mismatch repair.

DNA bending and unbending by MutS govern mismatch recognition and specificity

DNA mismatch repair is central to the maintenance of genomic stability. It is initiated by the recognition of base–base mismatches and insertion/deletion loops by the family of MutS proteins. Subsequently, ATP induces a unique conformational change in the MutS–mismatch complex but not in the MutS–homoduplex complex that sets off the cascade of events that leads to repair. To gain insight into the mechanism by which MutS discriminates between mismatch and homoduplex DNA, we have examined the conformations of specific and nonspecific MutS–DNA complexes by using atomic force microscopy. Interestingly, MutS–DNA complexes exhibit a single population of conformations, in which the DNA is bent at homoduplex sites, but two populations of conformations, bent and unbent, at mismatch sites. These results suggest that the specific recognition complex is one in which the DNA is unbent. Combining our results with existing biochemical and crystallographic data leads us to propose that MutS: (i) binds to DNA nonspecifically and bends it in search of a mismatch; (ii) on specific recognition of a mismatch, undergoes a conformational change to an initial recognition complex in which the DNA is kinked, with interactions similar to those in the published crystal structures; and (iii) finally undergoes a further conformational change to the ultimate recognition complex in which the DNA is unbent. Our results provide a structural explanation for the long-standing question of how MutS achieves mismatch repair specificity.

Parallel DNA triplexes, homologous recombination, and other homology-dependent DNA interactions

R. Daniel Camerini-Otero and Peggy Hsieh Genetics and Biochemistry Branch National institute of Diabetes and Digestive and Kidney Diseases National institutes of Health Bethesda, Maryland 20892 Genetic recombination is the process, common to all forms of life, by which new combinations of genetic material or nucleic acid sequences are generated (for a historical per- spective see Whitehouse, 1982). Biologists have been fas- cinated with this phenomenon for almost a century. In 1905, while studying the inheritance of traits in the sweet pea (Lathyrus odoratus), Bateson and colleagues reported an exception to Mendel’s third law, that of independent segregation. Certain combinations of traits were observed more frequently and others less frequently than expected. The mechanism underlying this partial linkage (neither complete linkage nor independent segregation) is recom- bination. Morgan coined the term “crossing over” to ex- plain the exchange that gave rise to new combinations of linked traits. This recombination involving exchanges of genetic information at equivalent positions anywhere along the length of two chromosomes with substantial overall sequence identity is called general or homologous recombination. A remarkable feature of this process is that, in spite of its lack of specificity, it has exquisite fidelity. That is, in homologous recombination, there is neither the loss nor the gain of a single nucleotide at the joint. These joints are therefore quite unlike the imprecise joints observed, for example, between coding regions in V(D)J recombina- tion. In a second class of recombination, site-specific re- combination, the two chromosomes exchange information in a very precise manner at sites of which at least one is highly preferred, or specific (Craig, 1988). In this form of recombination, overall homology between the two chro- mosomes is not a factor. It is curious that mechanistically we know much more about site-specific recombination than about general re- combination. To a great extent, this disparity in our state of knowledge is a reflection of how much more amenable site-specific recombination is to biochemical dissection. For example, the reactions are well defined; that is, the biologically relevant substrates and products can be easily distinguished biochemically, and only a very few proteins are involved (one in some cases). Perhaps as important, most and usually all of the proteins involved are encoded by the smaller of the substrate DNAs, the largest of which is the size of a bacteriophage genome. As a consequence, the first of many complete site-specific recombination re- actions was achieved in a cell-free system over 15 years ago (Craig, 1988). These assays were then used to purify all the components involved in several of these reactions. By contrast, in meiotic general recombination, the de- tails are so elaborate that they border on the rococo. Even in bacteria (during conjugation or transduction, for exam- ple), the details of homologous recombination are quite daunting, and the reaction(s) is hard to define. First, by definition the substrates and products are virtually indistin- guishable. This rather featureless aspect of homologous recombination still remains a great stumbling block in es- tablishing the relevance of in vitro biochemical findings to events in cells. Second, in Escherichia coli at least 20 gene products are known that participate in three separa- ble pathways of general recombination (Smith, 1989). Almost as important, there is a fundamental mechanistic difference between these two forms of recombination that follows from the difference in specificity. In site-specific recombination, specific DNA sequences are brought to- gether by protein-protein bridges anchored on specific protein-DNA complexes. In essence, the paradigm used for molecular recognition is not unlike that used in all other specific DNA-protein interactions. That is, evolution has sculpted protein surfaces that recognize certain features usually present in the major groove of duplex DNA. In contrast, in general (homologous) recombination, a similar degree of precision, and in some cases an even greater degree of fidelity, has to be achieved in the absence sequence specificity. Thus, the biochemical machinery cannot draw on such carefully crafted specific protein- DNA interactions. This has been the alluring mechanistic challenge in understanding general recombination: how are any two homologous DNA sequences brought to- gether? An early idea for fidelity of homologous recombina- tion was the copy choice model, which postulated DNA template switching during replication. In this model, it was assumed that some unspecified previous pairing of the chromosomes brought them into sufficiently close proxim- ity that accurate switching between DNA duplexes would ensue. The break-and-join paradigm, based on the ob- served behavior of chromosomes at meiosis, was consid- ered to be too imprecise account for the fidelity of this kind of genetic exchange. Although in hindsight it appears obvious that the required fidelity in the face of nonspeci- ficity could take advantage of direct DNA-DNA interac-

Somatic deletions of genes regulating MSH2 protein stability cause DNA mismatch repair deficiency and drug resistance in human leukemia cells

DNA mismatch repair enzymes (for example, MSH2) maintain genomic integrity, and their deficiency predisposes to several human cancers and to drug resistance. We found that leukemia cells from a substantial proportion of children (∼11%) with newly diagnosed acute lymphoblastic leukemia have low or undetectable MSH2 protein levels, despite abundant wild-type MSH2 mRNA. Leukemia cells with low levels of MSH2 contained partial or complete somatic deletions of one to four genes that regulate MSH2 degradation (FRAP1 (also known as MTOR), HERC1, PRKCZ and PIK3C2B); we also found these deletions in individuals with adult acute lymphoblastic leukemia (16%) and sporadic colorectal cancer (13.5%). Knockdown of these genes in human leukemia cells recapitulated the MSH2 protein deficiency by enhancing MSH2 degradation, leading to substantial reduction in DNA mismatch repair and increased resistance to thiopurines. These findings reveal a previously unrecognized mechanism whereby somatic deletions of genes regulating MSH2 degradation result in undetectable levels of MSH2 protein in leukemia cells, DNA mismatch repair deficiency and drug resistance.

Mechanism of MutS Searching for DNA Mismatches and Signaling Repair

DNA mismatch repair is initiated by the recognition of mismatches by MutS proteins. The mechanism by which MutS searches for and recognizes mismatches and subsequently signals repair remains poorly understood. We used single-molecule analyses of atomic force microscopy images of MutS-DNA complexes, coupled with biochemical assays, to determine the distributions of conformational states, the DNA binding affinities, and the ATPase activities of wild type and two mutants of MutS, with alanine substitutions in the conserved Phe-Xaa-Glu mismatch recognition motif. We find that on homoduplex DNA, the conserved Glu, but not the Phe, facilitates MutS-induced DNA bending, whereas at mismatches, both Phe and Glu promote the formation of an unbent conformation. The data reveal an unusual role for the Phe residue in that it promotes the unbending, not bending, of DNA at mismatch sites. In addition, formation of the specific unbent MutS-DNA conformation at mismatches appears to be required for the inhibition of ATP hydrolysis by MutS that signals initiation of repair. These results provide a structural explanation for the mechanism by which MutS searches for and recognizes mismatches and for the observed phenotypes of mutants with substitutions in the Phe-Xaa-Glu motif.

INTERACTION OF MUTS PROTEIN WITH THE MAJOR AND MINOR GROOVES OF A HETERODUPLEX DNA

Thermus aquaticus MutS protein is a DNA mismatch repair protein that recognizes and binds to heteroduplex DNAs containing mispaired or unpaired bases. Using enzymatic and chemical probe methods, we have examined the binding of TaqMutS protein to a heteroduplex DNA having a single unpaired thymidine residue. DNase I footprinting identifies a symmetrical region of protection 24–28 nucleotides long centered on the unpaired base. Methylation protection and interference studies establish thatTaq MutS protein makes contacts with the major groove of the heteroduplex in the immediate vicinity of the unpaired base. Hydroxyl radical and 1,10-phenanthroline-copper footprinting experiments indicate that MutS also interacts with the minor groove near the unpaired base. Together with the identification of key phosphate groups detected by ethylation interference, these data reveal critical contact points residing in the major and minor grooves of the heteroduplex DNA.

The mismatch repair-mediated cell cycle checkpoint response to fluorodeoxyuridine.

The loss of DNA mismatch repair (MMR) is responsible for hereditary nonpolyposis colorectal cancer and a subset of sporadic tumors. Acquired resistance or tolerance to some anti‐cancer drugs occurs when MMR function is impaired. 5‐Fluorouracil (FU), an anti‐cancer drug used in the treatment of advanced colorectal and other cancers, and its metabolites are incorporated into RNA and DNA and inhibit thymidylate synthase resulting in depletion of dTTP and incorporation in DNA of uracil. Although the MMR deficiency has been implicated in tolerance to FU, the mechanism of cell killing remains unclear. Here, we examine the cellular response to fluorodeoxyuridine (FdU) and the role of the MMR system. After brief exposure of cells to low doses of FdU, MMR mediates DNA damage signaling during S‐phase and triggers arrest in G2/M in the first cell cycle in a manner requiring MutSα, MutLα, and DNA replication. Cell cycle arrest is mediated by ATR kinase and results in phosphorylation of Chk1 and SMC1. MutSα binds FdU:G mispairs in vitro consistent with its being a DNA damage sensor. Prolonged treatment with FdU results in an irreversible arrest in G2 that is independent of MMR status and leads to the accumulation of DNA lesions that are targeted by the base excision repair (BER) pathway. Thus, MMR can act as a direct sensor of FdU‐mediated DNA lesions eliciting cell cycle arrest via the ATR/Chk1 pathway. However, at higher levels of damage, other damage surveillance pathways such as BER also play important roles. J. Cell. Biochem. 105: 245–254, 2008.

DNA mismatch repair and the DNA damage response.

This review discusses the role of DNA mismatch repair (MMR) in the DNA damage response (DDR) that triggers cell cycle arrest and, in some cases, apoptosis. Although the focus is on findings from mammalian cells, much has been learned from studies in other organisms including bacteria and yeast [1,2]. MMR promotes a DDR mediated by a key signaling kinase, ATM and Rad3-related (ATR), in response to various types of DNA damage including some encountered in widely used chemotherapy regimes. An introduction to the DDR mediated by ATR reveals its immense complexity and highlights the many biological and mechanistic questions that remain. Recent findings and future directions are highlighted.