Structural Studies Of DNA Recombination, Repair, and Rep

Diabetes, Digestive, Kidney Diseases

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Abstract

DNA is susceptible to a variety of mutations and chemical modifications. Errors during DNA replication, either mispairing or slippage, result in mismatched base pairs, which occur at a frequency of 10-8 to 10-6. Exposure to UV irradiation or chemical agents may lead to covalently modified DNA bases, and programmed meiotic and mitotic DNA rearrangement, ionizing radiation and oxidative agents can result in double-strand DNA breaks. To maintain genomic integrity and to sustain life, bacteria, archaea and eukarya use conserved mechanisms to repair or to tolerate each type of damage. My research group has continued to carry on structural and functional studies of E. coli and human mismatch repair processes and lesion-bypass DNA synthesis. Mismatch repair (MMR) in E. coli is initiated by three proteins, MutS, MutL and MutH, to specifically target the newly synthesized daughter strand. MutS is an ATPase and recognizes a mismatched base-pair as well as an insertion or deletion of 1-4 nucleotides in one strand. MutH is a latent endonuclease that is both sequence- and methylation-specific; when activated by MutS upon detection of a mismatch, it cleaves 5? to the unmethylated d(GATC) sequence in a hemimethylated duplex. MutL mediates the communication between MutS and MutH, which do not directly interact. Once a nick is introduced to the daughter strand by MutH, UvrD helicase, single-strand binding protein and DNA exonuclease, UvrD are recruited to remove nucleotides from the nick to beyond the mismatch. Homologues of MutS and MutL are found in all eukaryotes, and malfunction of either human MutS or MutL homolog is directly implicated in the susceptibility to hereditary non-polyposis colorectal cancer (HNPCC) and other sporadic cancers. Our previous studies suggest that the broad range of mismatch-repair substrates and high repair specificity are achieved with the high energy factor, ATP, utilized by MutS to verify and proofread mismatch recognition and to recruit MutL to signal for repair. In this year, we have determined the crystal structure of the C-terminal dimerization domain of MutL, characterized its DNA-binding and protein-interacting role in MMR. Based on out biochemical and genetic data, we propose a model that explains how the strand nicking (1st step) occurs either 5' or 3' to the mismatch site and the strand removal (2nd step by UvrD and exonucleases) is usually directed towards the mismatch site (Guarne et al, 2004). In summary, we have attained a complete working model of mismatch repair. In collaboration with Steven Lipkin at UC Irvine, we have also combine the population genetics, biochemistry and structural biology to analyze increased susceptibility to colorectal cancer in Israeli individuals due to a point mutation and attenuated activity of human MutL homolog 1 (MLH1) (Lipkin et al., 2004). Lesion-bypass DNA synthesis is carried out by the recently discovered Y-family DNA polymerases, which perform low-fidelity synthesis on undamaged DNA templates and are able to traverse normally replication-blocking lesions, including abasic sites, 8-oxo-G, benzopyrene adducts, and cyclobutane pyrimidine dimers. Y-family polymerases are widespread and enable species from E. coli to human to tolerate UV irradiation and various forms of base modification. Each individual Y-family polymerase exhibits a distinct substrate preference. For example, Pol h is particularly efficient to bypass the UV crosslinking product, cyclobutane pyrimidine dimers. Mutations in XPV, which encodes human Pol h, are correlated to 20% of xeroderma pigmentosum. After publishing the first Y-family polymerase and DNA complex structure in 2001 and the crystal structures of Dpo4 complexed with a cyclobutane pyrimidine dimers in 2003, this year we have reported a crystal structure of Dpo4 complexed with a benzo[a]pyrene adduct (Ling et al., 2004A) and five structures of the polymerase complexed wwwith abasic lesion in various reaction states (Ling et al., 2004B). Our structures suggest a mechanism by which specific Y-family polymerases are able to bypass a benzo[a]pyrene adduct or abasic lesion, while replicative DNA polymerases cannot. We are continuing to study structure of multiprotein and nucleic acid complexes involving in DNA repair and replication. Our goal is to elucidating molecular mechanism that underlies in human diseases.