PARENT SESSION
9:00 AM to 11:00 AM
Sunday, April 21, 2002
Symposium 4
Genes Affecting Radiosensitivity and Genomic Instability: From Yeast to Man
Room: Crystal Ballroom 1-2
Chair: Brown, J. Martin 11Stanford University, Stanford, CA
Speakers: Nickloff, Jac2; Brown, J. Martin1; Thompson, Larry42University of New Mexico School of Medicine, Albuquerque, NM1Stanford University, Stanford, CA41069 Felicia Ct., Livermore, CA, US
(S04-3) Role of the Rad51 paralogs in radiation sensitivity and genome stability.
Thompson, Larry*,1, Liu, Nan1, 1 Biol. & Biotech. Res. Program, L441, Livermore, CA
ABSTRACT-
Double-strand breaks (DSBs) are repaired by the pathways of nonhomologous end joining and homologous recombinational repair (HRR). In HRR the central player is Rad51, which forms nucleoprotein filaments that promote homologous pairing and strand exchange. In vertebrates, the strand-exchange activity of Rad51 requires other factors, including five proteins referred to as Rad51 paralogs (XRCC2, XRCC3, Rad51B, Rad51C, and Rad51D). These paralogs are highly diverged from both Rad51 and from each other (20-30% pairwise identities), indicating that each protein has evolved a new function. The phenotypes of rodent and chicken cell mutants of these paralogs are all very similar and include greatly increased spontaneous chromosomal aberrations, increased sensitivity to killing by various DNA damaging agents, lack of Rad51 nuclear focus formation in response to damage, and decreased efficiency of gene targeting. Whereas the loss of a Rad51 paralog reduces the growth rate and plating efficiency, a null mutation for Rad51 is fully lethal, implying that a residual level of HRR remains in Rad51 paralog mutants. Analyses of protein interactions in cell extracts indicate that the paralogs exist in at least two discrete complexes, a Rad51C-XRCC3 heterodimer and a Rad51B-Rad51C-Rad51D-XRCC2 complex (Liu et al. NAR, in press). In an effort to create additional CHO mutants for the paralogs, we performed gene knockout at the RAD51C locus, and disrupted one allele with an efficiency of targeting of <10E-3 among G418-resistant transformants. Work is in progress to recover cells in which the second allele has been inactivated. To assess the contribution of XRCC3 during the cell cycle to survival after g-irradiation, we synchronized irs1SF and wild-type AA8 CHO cells by centrifugal elutriation. AA8 cells exposed to 6 Gy (survival = 13%) were most sensitive in early G1 and most resistant during S phase. Irs1SF cells were treated with 3 Gy to produce 10% survival. The pattern of sensitivity for irs1SF was reversed compared with that of AA8, with S phase being the most sensitive. Thus, HRR appears to be critically important during the S and G2 phases of the cell cycle when a sister chromatid is available to serve as a template for DSB repair. (Work was done under the auspices of the US DOE by LLNL under contract No. W-7405-ENG-48).
KEYWORDS: genomic instability, Rad51 paralogs, chromosomal aberrations, cell cycle response to radiation