Analysis of DNA mismatch repair in cellular response to DNA damage.
Significant advances have been made in identifying and characterizing the roles of DNA mismatch repair (MMR) proteins in cellular response to DNA damage. Insights into this process have been obtained by performing interactions of mismatch recognition proteins (e.g., MutSalpha) with DNA adduct-containing duplexes and by analyzing cellular responses (including cell cycle checkpoints and apoptosis) of cell lines and animals with various MMR capacities. This chapter presents detailed methods for gel-shift analysis to determine the interaction between MutSalpha and oligonucleotide duplex containing a single DNA adduct and for apoptotic assays in cell lines and experimental animals. In addition, a step-by-step protocol is also provided for the purification of MutSalpha from human cells, the preparation of DNA substrates containing a defined DNA adduct, and the treatment of MMR-proficient and deficient cell lines as well as MMR knockout mice.
Gu
Liya
L
Department of Toxicology and Pathology, University of Kentucky Medical Center, Lexington, USA.
Li
Guo-Min
GM
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D017209:Apoptosis; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D018736:DNA Adducts; D004249:DNA Damage; D053938:DNA Fragmentation; D053843:DNA Mismatch Repair; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D000070957:MutL Protein Homolog 1; D051719:MutS DNA Mismatch-Binding Protein / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018736:DNA Adducts; C501485:Mlh1 protein, mouse; D009687:Nuclear Proteins; D021122:Protein Subunits; D004247:DNA; D000070957:MutL Protein Homolog 1; D051719:MutS DNA Mismatch-Binding Protein