Introduction
DNA mismatch repair plays a crucial role in the maintenance of genomic stability. In E. coli, the repair process is initiated by the binding of a MutS homodimer to mismatches or unpaired nucleotides that arose as errors of DNA polymerase and escaped the proofreading activity of the DNA replication machinery, or during recombination between homologous but non-identical DNA sequences. Mismatch binding triggers an ATP-dependent conformational change in MutS, which then recruits a MutL homodimer. The formation of this ternary complex subsequently activates a cascade of events that involve the recruitment of the strand-discrimination endonuclease MutH, DNA helicase II (UvrD), one of several exonucleases, DNA polymerase III and DNA ligase.
The main features of the DNA mismatch repair system are conserved from E. coli to man. In eukaryotes, the MutS function is mediated by MutS homologues (MSH); the heterodimer of MSH2 and MSH6 (MutSα) is mainly involved in the recognition of base-base mismatches, while MutSα (a heterodimer of MSH2 and MSH3) is primarily involved in the recognition of small insertion and deletion loops. Similarly, the eukaryotic MutL homologues function in the form of heterodimers; MutLα is composed of MLH1 and PMS2, MutLβ of MLH1 and PMS1, and MutLγ consists of MLH1 and MLH3. There is no MutH homologue in eukaryotes, but recent work suggests that the endonuclease activity resides in the MutL homologues.
In both bacteria and eukaryotes, the loss of mismatch repair gives rise to a mutator phenotype. In hereditary non-polyposis colon cancer (HNPCC) families, germline mutations in one MSH2, MSH6 or MLH1 allele predispose to cancer of the colon, endometrium, ovary and other organs. HNPCC is the most frequent form of familiar cancer. The young age of onset and high disease penetrance indicates that the loss of the wild type copy of the mutated gene occurs with high frequency in epithelial cells. Moreover, about 10% of colon cancers display a phenotype identical to that of HNPCC tumours. This phenotype is not linked to MMR gene mutations; rather, it is associated with the epigenetic silencing of the MMR gene MLH1.
Mismatch repair is a highly-complex, but also a highly-efficient mechanism of DNA metabolism. It improves the fidelity of DNA replication by up to three orders of magnitude, which requires that its constitutive factors cooperate in a precisely defined manner. In particular, the steps of mismatch recognition and strand discrimination have to be strictly controlled, given that MMR has to be directed to the newly-synthesized DNA strand (in the case of replication) or to the invading DNA strand (in the case of recombination). How this is accomplished in the eukaryotic system is currently unclear. The E. coli system involves the MutH endonuclease, which is activated by the MutS/MutL complex to cleave the unmethylated strand at GATC sites in the newly-synthesised DNA, but it is not known how. Given that the strand discrimination signal can be as far as a kilobase from the mismatch and still direct the repair process, the molecular mechanism of this key signal transmission process is of substantial interest. Indeed, proposed mechanisms for the manner in which MutS, MutL and MutH structurally and biomechanically couple mismatch recognition to strand discrimination vary broadly, from sliding clamp mechanisms to translocating molecular motors. Clearly, the structural and mechanistic details that govern this process are crucial to identifying and understanding the system's rate-limiting features.
In recent years, we have been able to acquire considerable wealth of knowledge about the eukaryotic MMR system. This work culminated in the reconstitution of the human system from purified recombinant constituent proteins. However, as in the case of E. coli, we currently lack the basic mechanistic insights into mismatch repair. In this proposal, we plan to use systems biology to learn about the rules that govern the principal steps of MMR: mismatch recognition, repairosome assembly, strand discrimination and strand degradation.
At the present time, the term “systems biology” is most often applied to describe high throughput proteomic studies that attempt to construct protein interaction maps. While these networks contain extremely valuable information, they do not tell us when these protein complexes form, when they disassemble and how these events are regulated. This information is, however, vital to our understanding of the functioning of a cell. Addition of the dynamics and regulation of protein complex formation to the existing interaction networks requires acquisition of affinity constants for the protein partners in the complexes, rates of association and dissociation, and information about the factors influencing these parameters.
In an attempt to reach the next level of understanding of complex biological processes, we now propose to study the individual steps of the pro- and eukaryotic mismatch repair process in fine detail, using a combination of high-resolution structural and kinetic experimental approaches with in vitro repair assays and mathematical modelling. This will involve single-molecule force spectroscopy and imaging (DNA nanomanipulation, single-molecule fluorescence energy transfer (FRET), scanning force microscopy (SFM) imaging), surface plasmon resonance imaging (SPR), structural biology (X-ray elucidation of DNA/protein and protein/protein complexes of increasing complexity, crosslinking studies) and mathematical modelling of first the kinetics and energetics of the latter processes, and, second, of the in vivo characteristics of MMR and its interactions with other DNA repair pathways. Thus, this consortium will generate, integrate and organize mechanistic information on a poorly-understood, yet indispensable process of DNA metabolism in both structural and kinetic detail, from the atomic level to the macromolecular and cellular levels. Such an approach at multiple levels is unique and will provide a prototype for other cellular processes.