TY - JOUR
T1 - Measuring strand discontinuity-directed mismatch repair in yeast Saccharomyces cerevisiae by cell-free nuclear extracts
AU - Yuan, Fenghua
AU - Lai, Fangfang
AU - Gu, Liya
AU - Zhou, Wen
AU - Hokayem, Jimmy El
AU - Zhang, Yanbin
N1 - Funding Information:
This research was supported, in part, by the Stanley J. Glaser Foundation and the Florida Bankhead-Coley Cancer Research Program.
Copyright:
Copyright 2009 Elsevier B.V., All rights reserved.
PY - 2009/5
Y1 - 2009/5
N2 - Mismatch repair corrects biosynthetic errors generated during DNA replication, whose deficiency causes a mutator phenotype and directly underlies hereditary non-polyposis colorectal cancer and sporadic cancers. Because of remarkably high conservation of the mismatch repair machinery between the budding yeast (Saccharomyces cerevisiae) and humans, the study of mismatch repair in yeast has provided tremendous insights into the mechanisms of this repair pathway in humans. In addition, yeast cells possess an unbeatable advantage over human cells in terms of the easy genetic manipulation, the availability of whole genome deletion strains, and the relatively low cost for setting up the system. Although many components of eukaryotic mismatch repair have been identified, it remains unclear if additional factors, such as DNA helicase(s) and redundant nuclease(s) besides EXO1, participate in eukaryotic mismatch repair. To facilitate the discovery of novel mismatch repair factors, we developed a straightforward in vitro cell-free repair system. Here, we describe the practical protocols for preparation of yeast cell-free nuclear extracts and DNA mismatch substrates, and the in vitro mismatch repair assay. The validity of the cell-free system was confirmed by the mismatch repair deficient yeast strain (Δmsh2) and the complementation assay with purified yeast MSH2-MSH6.
AB - Mismatch repair corrects biosynthetic errors generated during DNA replication, whose deficiency causes a mutator phenotype and directly underlies hereditary non-polyposis colorectal cancer and sporadic cancers. Because of remarkably high conservation of the mismatch repair machinery between the budding yeast (Saccharomyces cerevisiae) and humans, the study of mismatch repair in yeast has provided tremendous insights into the mechanisms of this repair pathway in humans. In addition, yeast cells possess an unbeatable advantage over human cells in terms of the easy genetic manipulation, the availability of whole genome deletion strains, and the relatively low cost for setting up the system. Although many components of eukaryotic mismatch repair have been identified, it remains unclear if additional factors, such as DNA helicase(s) and redundant nuclease(s) besides EXO1, participate in eukaryotic mismatch repair. To facilitate the discovery of novel mismatch repair factors, we developed a straightforward in vitro cell-free repair system. Here, we describe the practical protocols for preparation of yeast cell-free nuclear extracts and DNA mismatch substrates, and the in vitro mismatch repair assay. The validity of the cell-free system was confirmed by the mismatch repair deficient yeast strain (Δmsh2) and the complementation assay with purified yeast MSH2-MSH6.
KW - Cancer
KW - Cell-free nuclear extract
KW - In vitro mismatch repair
KW - MMR
KW - Saccharomyces cerevisiae
KW - Yeast
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U2 - 10.1016/j.ymeth.2009.02.014
DO - 10.1016/j.ymeth.2009.02.014
M3 - Article
C2 - 19250969
AN - SCOPUS:65649086948
SN - 1046-2023
VL - 48
SP - 14
EP - 18
JO - Methods
JF - Methods
IS - 1
ER -