Structural and mechanistic studies Of DNA mismatch repair

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Mismatch repair (MMR) in E. coli is initiated by three proteins, MutS, MutL and MutH to specifically target 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 is also an ATPase and 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 are recruited by MutS and MutL 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 led to the determination of crystal structures of MutS, MutS-mismatch DNA and MutS-mismatch-ADP complexes, the N- and C-terminal domain of MutL, and finally MutH and MutH-DNA complexes. We also characterized the role of the MutS and MutL ATPases and the cleavage specificity of MutH. In this fiscal year, we have succeeded in determining a series of crystal structures of UvrD helicase-DNA complexes, which represent consecutive physical steps of UvrD unwinding a duplex DNA in an ATP hydrolysis cycle. In addition, we have carried out mutagenesis studies to dissect two alternative mechanisms of DNA unwinding by UvrD. Our manuscript UvrD helicase unwinds DNA one base pair at a time by a two-part power stroke was published in Cell in December 2006. Currently we are engaging in (1) obtaining large protein-DNA assemblies, e.g. MutL-DNA, MutL-UvrD-DNA, MutS-MutL-DNA complexes, for structural characterization, (2) pre-steady state kinetic studies of the ATPases involved in mismatch repair, and (3) expanding structural and mechanistic studies to eukaryotic mismatch repair systems. In the previous year (2021-22), Dr. Jun Li has made two breakthrough in the mismatch repair by successfully determining cryoEM structure of MutS-DNA complex in the presence of ATP and ATP analogs to understand the "signal transduction" process and by characterization of how human MutSbeta promotes trinucleotide repeats, such as CTG, CAG, CCG and CGG to expand, which lead to various neurological diseases, including Huntington Disease, myotonic dystrophy and fragile X syndrome. We expect that our findings in these two areas both lead to paradigm-shift publications in the coming years. References Ortega, J., Lee, G.S., Gu, L., Yang, W. & Li, G.M. (2021) Mispair-bound human MutS-MutL complex triggers DNA incisions and activates mismatch repair. Cell Research, 31, 542-553. Gu, H. Z., Yang, W. & Seeman, N. C. (2010). DNA scissors device used to measure MutS binding to DNA mis-pairs, JACS, 132, p4252-4357. Yang, W. (2010) Lessons learnt from UvrD helicase: mechanism for directional movement. Ann. Rev. Biophys., 39, 367-385.