Publication Date

5-2018

Advisor(s)

Ishita Mukerji

Department

Molecular Biology & Biochemistry

Abstract

The DNA mismatch repair (MMR) system guards the integrity of genome by scanning and correcting errors in a post-replicative manner. In eukaryotes, the initiation of MMR is achieved by Msh2-Msh6 heterodimer which recognizes single base mismatches and small insertion/deletion loops. Although Msh2-Msh6 recognizes mismatched DNA with high affinity, the exact mechanism by which Msh2-Msh6 distinguishes different types of mismatched base pairs (bp) from canonical Watson-Crick bps is still unknown. In this study, we use the intrinsic fluorescent probe 6-methylisoxanthopterin (6-MI, guanosine analogue) in the context of the ATFAA (F = 6-MI) pentamer sequence where it exhibits enhanced fluorescence, to measure the binding affinity of S. cerevisiae Msh2-Msh6 to different single bp mismatches. Fluorescence intensity and Förster resonance energy transfer measurements suggest DNA distortion accompanies binding to mismatched bp. We have also investigated DNA dynamics upon Msh2-Msh6 binding using time-resolved fluorescence spectroscopy. Specific placement of the probe at the mismatch site or adjacent to it reveals significant local motion prior to protein binding. We observe that high affinity binding is associated with those mismatches that exhibit the greatest amount of motion. A directional stabilization at the mismatch site or 3’ to the mismatch is observed upon Msh2-Msh binding, which is consistent with Phe intercalation at the site, as observed in Msh2-Msh6-DNA co-crystal structures.

Holliday junctions (HJ) are four-stranded DNA structures that are functional intermediates observed in recombination and MMR. We have confirmed using gel mobility shift assay and fluorescence spectroscopy that S. cerevisiae Msh2-Msh6 binds to HJ with nanomolar affinity comparable to that observed for mismatched duplex DNA. A protein:DNA binding stoichiometry of 1:1 is determined for Msh2-Msh6 and HJ. Furthermore, the steady state ATPase activity of Msh2-Msh6 indicates that ATP hydrolysis was only slightly stimulated by HJ binding similar to mismatched DNA.

Eukaryotic Msh4-Msh5 heterodimer mainly functions in promoting the formation of meiotic crossovers in Meiosis I. To further report on the specific binding of S. cerevisiae Msh4-Msh5 with the recombination intermediate, and to further detect its binding parameters with other DNA substrates, we cloned msh4 and msh5 genes from yeast genomic DNA for over-expression in E. coli.

Available for download on Tuesday, June 01, 2021

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