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Biochemical Analysis of S. cerevisiae Msh2‐Msh3 Activity in Triplet Nucleotide Repeat Expansion
Author(s) -
Yu Xiaoyu Zoë,
Sawant Shreya,
Hingorani Manju
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.542.3
Subject(s) - dna mismatch repair , msh2 , base pair , nucleotide , dna , biology , msh6 , chemistry , microbiology and biotechnology , dna repair , genetics , biophysics , gene
Mismatch repair mechanisms improve DNA replication fidelity to only one base‐pairing error in 10 9 nucleotides. Msh2‐Msh6 (MutSα) is responsible for initiating repair of single base mismatches and small insertion/deletion loops and Msh2‐Msh3 (MutSβ) is responsible for insertion/deletion loops. However, Msh2‐Msh3 can also stabilize loops/hairpins formed by repeat nucleotide sequences and contribute to triplet nucleotide repeat (TNR) expansion. The goal of this study is to investigate the S.cerevisiae Msh2‐Msh3 mechanism of action by transient kinetics, in order to understand how it can accomplish both loop repair and expansion. Thus far, y Msh2‐Msh3 protein complex has been expressed in E.coli host cells and can be purified in large quantity for biochemical studies. I am optimizing the purification protocol by testing different ion exchange and affinity exchange chromatography resins. I am also developing different DNA substrates to assay the affinity and kinetics of y Msh2‐Msh3 binding to loops/hairpins and its coupled ATPase activity. This study is expected to yield kinetic parameters that distinguish between y Msh2‐Msh3 actions that trigger loop repair versus TNR expansion.