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RAD54 family translocases counter genotoxic effects of RAD51 in human tumor cells.

Mason JM, Dusad K, Wright WD, Grubb J, Budke B, Heyer WD, Connell PP, Weichselbaum RR, Bishop DK - Nucleic Acids Res. (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation and Cellular Oncology, University of Chicago, Cummings Life Science Center, Box 13, 920 East 58th St., Chicago, IL 60637, USA.

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Depletion of translocases does not result in an increase in markers of DNA damage. Representative images of nuclei containing RAD51 foci and either (a) 53BP1 foci (b) γ-H2AX foci or (c) PCNA. (d-g) Dot plots representing the number of foci in MCF7 cells after the indicated treatments for d) RAD51, (e) 53BP1 (f) gH2AX (g) and RAD51 in PCNA-negative and PCNA-positive nuclei. Red lines and numbers above the graphs represent the mean RAD51 focus counts. Scale bar; 5 μm.
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Figure 3: Depletion of translocases does not result in an increase in markers of DNA damage. Representative images of nuclei containing RAD51 foci and either (a) 53BP1 foci (b) γ-H2AX foci or (c) PCNA. (d-g) Dot plots representing the number of foci in MCF7 cells after the indicated treatments for d) RAD51, (e) 53BP1 (f) gH2AX (g) and RAD51 in PCNA-negative and PCNA-positive nuclei. Red lines and numbers above the graphs represent the mean RAD51 focus counts. Scale bar; 5 μm.

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.


RAD54 family translocases counter genotoxic effects of RAD51 in human tumor cells.

Mason JM, Dusad K, Wright WD, Grubb J, Budke B, Heyer WD, Connell PP, Weichselbaum RR, Bishop DK - Nucleic Acids Res. (2015)

Depletion of translocases does not result in an increase in markers of DNA damage. Representative images of nuclei containing RAD51 foci and either (a) 53BP1 foci (b) γ-H2AX foci or (c) PCNA. (d-g) Dot plots representing the number of foci in MCF7 cells after the indicated treatments for d) RAD51, (e) 53BP1 (f) gH2AX (g) and RAD51 in PCNA-negative and PCNA-positive nuclei. Red lines and numbers above the graphs represent the mean RAD51 focus counts. Scale bar; 5 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4381078&req=5

Figure 3: Depletion of translocases does not result in an increase in markers of DNA damage. Representative images of nuclei containing RAD51 foci and either (a) 53BP1 foci (b) γ-H2AX foci or (c) PCNA. (d-g) Dot plots representing the number of foci in MCF7 cells after the indicated treatments for d) RAD51, (e) 53BP1 (f) gH2AX (g) and RAD51 in PCNA-negative and PCNA-positive nuclei. Red lines and numbers above the graphs represent the mean RAD51 focus counts. Scale bar; 5 μm.
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.

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.

View Article: PubMed Central - PubMed

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
Related in: MedlinePlus