Measuring strand discontinuity-directed mismatch repair in yeast Saccharomyces cerevisiae by cell-free nuclear extracts.
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 (Deltamsh2) and the complementation assay with purified yeast MSH2-MSH6.
Yuan
Fenghua
F
Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, FL 33136, USA.
Lai
Fangfang
F
Gu
Liya
L
Zhou
Wen
W
El Hokayem
Jimmy
J
Zhang
Yanbin
Y
D002467:Cell Nucleus / Q000378:metabolism; D002474:Cell-Free System / Q000378:metabolism; D053843:DNA Mismatch Repair / Q000235:genetics*; D004260:DNA Repair; D004271:DNA, Fungal / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D005816:Genetic Complementation Test; D051718:MutS Homolog 2 Protein / Q000378:metabolism; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; C094443:G-T mismatch-binding protein; D029701:Saccharomyces cerevisiae Proteins; D005090:Exodeoxyribonucleases; C028450:exodeoxyribonuclease I; D051718:MutS Homolog 2 Protein