BCCIP regulates homologous recombination by distinct domains and suppresses spontaneous DNA damage

Abstract

Homologous recombination (HR) is critical for maintaining genome stability through precise repair of DNA double-strand breaks (DSBs) and restarting stalled or collapsed DNA replication forks. HR is regulated by many proteins through distinct mechanisms. Some proteins have direct enzymatic roles in HR reactions, while others act as accessory factors that regulate HR enzymatic activity or coordinate HR with other cellular processes such as the cell cycle. The breast cancer susceptibility gene BRCA2 encodes a critical accessory protein that interacts with the RAD51 recombinase and this interaction fluctuates during the cell cycle. We previously showed that a BRCA2- and p21-interacting protein, BCCIP, regulates BRCA2 and RAD51 nuclear focus formation, DSB-induced HR and cell cycle progression. However, it has not been clear whether BCCIP acts exclusively through BRCA2 to regulate HR and whether BCCIP also regulates the alternative DSB repair pathway, non-homologous end joining. In this study, we found that BCCIP fragments that interact with BRCA2 or with p21 each inhibit DSB repair by HR. We further show that transient down-regulation of BCCIP in human cells does not affect non-specific integration of transfected DNA, but significantly inhibits homology-directed gene targeting. Furthermore, human HT1080 cells with constitutive down-regulation of BCCIP display increased levels of spontaneous single-stranded DNA (ssDNA) and DSBs. These data indicate that multiple BCCIP domains are important for HR regulation, that BCCIP is unlikely to regulate non-homologous end joining, and that BCCIP plays a critical role in resolving spontaneous DNA damage.

Figure 1.

BRCA2- and p21-interacting BCCIP fragments inhibit HR-mediated DSB repair. BCCIP and fragments were expressed from pCMV-Myc vector after transient transfection. The pCMV-myc empty vector was used as the negative control. (A) Maps of BCCIP fragments tested for effects on HR. (B) Intracellular distribution of myc-tagged BCCIP fragments detected by anti-Myc immunofluorescence microscopy. (C) Expression of myc-tagged BCCIP fragments detected by anti-myc western blot. (D) Relative frequencies of HR repair of DSBs in cells transiently expressing myc-tagged BCCIP fragments.

Figure 2.

Effects of BCCIP down-regulation and over-expression on gene targeting and random integration. Adenovirus vectors were used to achieve high efficiency transient BCCIP knockdown or over-expression in HT1080 cells. Control, cells infected with adenovirus expressing GFP alone; shRNA-BCCIP, cells infected with viruses expressing shRNA against both isoforms of BCCIP; HA-BCCIPα, cells infected with viruses expressing myc-tagged BCCIPα; myc-BCCIPβ, cells infected with viruses expressing myc-tagged BCCIPβ. (A) Gene targeting strategy. The HPRT locus is shown above and the pHRPT-hyg gene targeting vector is below. Targeted integration inserts the hygromycin resistance cassette into HPRT, conferring resistance to both hygromycin and 6TG (right). Random integration confers resistance only to hygromycin (left) (See text and reference 25 for more details). (B) Cell cycle distribution at the time when the gene targeting vector was electroporated (3 days after the virus infection). (C) Western blots demonstrating under- (left panel) and over- (right panel) expression of BCCIP. (D) BCCIP under- and over-expression does not affect random integration, determined as the total number of hygromycin-resistant (Hyg^R) colonies per viable cell. Values are averages ± standard error (SE). (E) BCCIP under-expression reduces gene targeting, determined as the number of Hyg^R and 6TG^R colonies per viable cell. Values are averages ± SE. (F) BCCIP under-expression reduces gene targeting, determined as the number of 6TG^R&Hyg^R colonies per Hyg^R colony. Values are averages ± SE.

Figure 3.

Expression of BCCIP-D and BCCIP-G fragments inhibit gene targeting. Adenovirus vectors were used to express: myc-BCCIP-D (aa 59–167), myc-BCCIP-G (aa168–258) and BRCA2-shRNA. Control cells were infected with adenovirus expressing GFP alone. Each group have total of 84 dishes for 6TGandHyg selection and were assayed in six independent experiments. (A) Western blots (3 days after the virus infection) confirming the expression of myc-BCCIP-D and myc-BCCIP-G (left panel), and the partial down-regulation of BRCA2 by BRCA2-shRNA (right panel). (B) Myc-BCCIP-D, myc-BCCIP-G and BRCA2-shRNA do not affect random integration, determined as the total number of hygromycin-resistant (Hyg^R) colonies per viable cell. Values are averages ± SE. For all groups compared with the control, the P-values were greater than 0.30 as tested by two tailed t-test. (C) Myc-BCCIP-D, Myc-BCCIP-G and BRCA2-shRNA reduce gene targeting, determined as the number of Hyg^R and 6TG^R colonies per viable cell. Values are averages ± SE. For all groups compared with the control, the P-values were less than 0.01 as tested by two tailed t-test. (D) Myc-BCCIP-D, Myc-BCCIP-G and BRCA2-shRNA reduce gene targeting, determined as the number of 6TG^R colonies per Hyg^R colony. Values are averages ± SE. For all groups compared with the control, the P-values were less than 0.01 as tested by two tailed t-test.

Figure 4.

Down-regulation of BCCIP causes high levels of spontaneous ssDNA. Cells with 2–3 weeks of constitutive partial BCCIP down-regulation were stained for ssDNA. Control cells were transfected with vectors expressing non-specific shRNA. In panels C–E, data are reported as means ± SD from three independent experiments. (A) Representative images of total DNA or ssDNA as visualized by anti-BrdU staining in control and partial BCCIP knockdown (BCCIP-KD) cells (see Materials and methods section for details on detecting total DNA and ssDNA). (B) The level of BCCIP in control and knockdown cells as detected by anti-BCCIP western blot (top panel). Level of actin (bottom panel) was used as loading control. (C) The percentage of cells with five or more ssDNA foci. The average percentage of cells with ≥5 foci were calculated from three independent experiments. For each experiment, at least 400 cells were counted for focus numbers. Statistical significance was evaluated using two-tailed t-test. (D) Integrated intensity of signals from ssDNA (stained with anti-BrdU without denaturing) and total DNA (stained with DAPI) in control and BCCIP-KD cells. (E) The relative amount of ssDNA as normalized to the total DNA in the cells.

Figure 5.

BCCIP knockdown cells show high levels of spontaneous γH2AX. Cells with 2–3 weeks of constitutive BCCIP down-regulation were stained by immunofluorescence microscopy using antibodies to γH2AX. (A) Representative nuclear staining of γH2AX in cells expressing control shRNA and BCCIP shRNA. DAPI staining of DNA indicates positions of nuclei in each field. (B) Average number of nuclear γH2AX foci per cell. Data are reported as mean ± SD from five independent experiments. For each experiment, at least 400 cells were counted for foci number. Statistical significance of γH2AX foci was analyzed by two-tailed t-test. (C) Total level of γH2AX protein in BCCIP knockdown cells was detected by anti-γH2AX Western blot (top panel). The level of endogenous BCCIP in the knockdown cells was detected by anti-BCCIP blot (middle panel). Level of actin was used as loading control.