Repression of mutagenesis by Rad51D-mediated homologous recombination.
Homologous recombinational repair (HRR) restores chromatid breaks arising during DNA replication and prevents chromosomal rearrangements that can occur from the misrepair of such breaks. In vertebrates, five Rad51 paralogs are identified that contribute in a nonessential but critical manner to HRR proficiency. We constructed and characterized a knockout of the paralog Rad51D in widely studied CHO cells. The rad51d mutant (clone 51D1) displays sensitivity to a diverse spectrum of induced DNA damage including gamma-rays, ultraviolet (UV)-C radiation, and methyl methanesulfonate (MMS), indicating the broad relevance of HRR to genotoxicity. Spontaneous chromatid breaks/gaps and isochromatid breaks are elevated 3- to 12-fold, but the chromosome number distribution remains unchanged. Most importantly, 51D1 cells exhibit a 12-fold-increased rate of hprt mutation, as well as 4- to 10-fold increased rates of gene amplification at the dhfr and CAD loci, respectively. Xrcc3 irs1SF cells from the same parental CHO line show similarly elevated mutagenesis at these three loci. Collectively, these results confirm the a priori expectation that HRR acts in an error-free manner to repress three classes of genetic alterations (chromosomal aberrations, loss of gene function and increased gene expression), all of which are associated with carcinogenesis.
Hinz
John M
JM
Biosciences Directorate, Lawrence Livermore National Laboratory, Livermore, CA 94551, USA.
Tebbs
Robert S
RS
Wilson
Paul F
PF
Nham
Peter B
PB
Salazar
Edmund P
EP
Nagasawa
Hatsumi
H
Urbin
Salustra S
SS
Bedford
Joel S
JS
Thompson
Larry H
LH
D000818:Animals; D016466:CHO Cells; D002470:Cell Survival; D002869:Chromosome Aberrations; D006224:Cricetinae; D003412:Cricetulus; D004249:DNA Damage; D005720:Gamma Rays; D005784:Gene Amplification; D018390:Gene Targeting; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D016296:Mutagenesis; D051135:Rad51 Recombinase / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D011995:Recombination, Genetic
D007041:Hypoxanthine Phosphoribosyltransferase; D051135:Rad51 Recombinase