Small-scale extracts for the study of nucleotide excision repair and non-homologous end joining.
The repair of DNA by nucleotide excision repair (NER) and non-homologous end joining (NHEJ) is essential for maintenance of genomic integrity and cell viability. Examination of NHEJ and NER in vitro using cell-free extracts has led to a deeper understanding of the biochemical mechanisms that underlie these processes. Current methods for production of whole-cell extracts (WCEs) to investigate NER and NHEJ start with one or more liters of culture containing 1-5 x 10(9) cells. Here, we describe a small-scale method for production of WCE that can be used to study NER. We also describe a rapid, small-scale method for the preparation of WCE that can be used in the study of NHEJ. These methods require less time, 20- to 1000-fold fewer cells than large-scale extracts, facilitate examination of numerous samples and are ideal for such applications as the study of host-virus interactions and analysis of mutant cell lines.
Smeaton
Michael B
MB
Department of Biochemistry and Molecular Biology, Johns Hopkins University, Bloomberg School of Public Health, Baltimore, MD 21205, USA.
Miller
Paul S
PS
Ketner
Gary
G
Hanakahi
Les A
LA
D000260:Adenoviruses, Human / Q000502:physiology; D000818:Animals; D016466:CHO Cells; D018929:Cell Culture Techniques; D002457:Cell Extracts / Q000235:genetics*; D002460:Cell Line; D002474:Cell-Free System; D006224:Cricetinae; D003412:Cricetulus; D004260:DNA Repair; D006367:HeLa Cells; D006801:Humans; D011995:Recombination, Genetic
D002457:Cell Extracts