RAD54 family translocases counter genotoxic effects of RAD51 in human tumor cells.
Bottom Line: We also show that translocase depletion in tumor cell lines leads to the accumulation of RAD51 on chromosomes, forming complexes that are not associated with markers of DNA damage.These results support a model in which RAD54L and RAD54B counteract genome-destabilizing effects of direct binding of RAD51 to dsDNA in human tumor cells.Thus, in addition to having genome-stabilizing DNA repair activity, human RAD51 has genome-destabilizing activity when expressed at high levels, as is the case in many human tumors.
Affiliation: Department of Radiation and Cellular Oncology, University of Chicago, Cummings Life Science Center, Box 13, 920 East 58th St., Chicago, IL 60637, USA.Show MeSH
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Mentions: To further support the conclusion that RAD51 foci observed in RAD54L+RAD54B depleted cells are not associated with DNA damage, we examined localization of RAD51 and two key DNA damage markers, 53BP1 and γ-H2AX. Both γ-H2AX and 53BP1 accumulate at DNA ends created by DSBs and collapsed replication forks (50–53). Translocase depletion resulted in a 2-fold increase in the average number of RAD51 foci/nucleus (4 ± 5 NS versus 8 ± 7 siRAD54L+RAD54B; P < 0.001; Figure 3a, d). However, RAD54L+RAD54B depletion did not increase the number of 53BP1 (4 ± 5 versus 4 ± 6 foci/nucleus) or γH2AX foci (5 ± 3 versus 7 ± 5 foci/nucleus) compared to controls (Figure 3 a,b,e,f). Furthermore, in RAD54L+B depleted cells, on average only 24 and 23% of RAD51 foci co-localized with 53BP1 and γH2AX, respectively. There is no significant difference in the number of 53BP1 and γH2AX foci between controls and translocase-depleted cells, so the co-localizing foci observed are unlikely to be as a result of increased spontaneous DNA damage. Both γH2AX and 53BP1 foci arise during S phase in unperturbed cells (54,55). Thus, the RAD51 foci in translocase-depleted cells that co-localize with DNA damage markers likely represent stalled/collapsed replication forks. In contrast, irradiation resulted in 3- and 5-fold increases in γH2AX and 53BP1 foci, respectively, and 83–86% of RAD51 foci exhibited co-localization with 53BP1 and γH2AX. This indicates that the majority of RAD51 foci that accumulate following translocase depletion in the absence of irradiation mark undamaged sites.
Affiliation: Department of Radiation and Cellular Oncology, University of Chicago, Cummings Life Science Center, Box 13, 920 East 58th St., Chicago, IL 60637, USA.