Fig. 1. BLM and p53 co-localize at stalled DNA replication forks in NHF. (A) BLM and p53 co-localized with BrdU. NHF were incubated with HU for 16 h, washed and incubation continued for 10 min in the presence of BrdU. Immunofluorescence was carried out with antibodies against (a) BrdU/BLM (C-18) and (b) BrdU/p53 (588). Quantitation of (a) and (b) is represented in (c) and (d). (B) BLM and p53 co-localized. NHF was treated with (a) HU or (b) PALA, and immunofluorescence was carried out with antibodies against BLM (C-18)/p53 (588). Quantitation of (a) and (b) is represented in (c) and (d). (C) Co-localization of p53, p53pSer15 and BLM. The same as (A) except that immunofluorescence was carried out with antibodies against (a) p53 (588)/p53pSer15 (16G8) and (c) BLM (C-18)/p53pSer15. Quantitation of (a) is represented in (b).

Fig. 7. Mechanistic model for the restoration of stalled DNA replication forks. A regressed replication fork is restored by either reverse branch migration mediated by BLM helicase in a non-recombinogenic pathway (indicated by a thick arrow, pathway A) or a recombinogenic pathway that involves endonucleolytic cleavage by a resolvase(s) (e.g. Mus81), followed by RAD51/RAD54-mediated HR (indicated by a thin arrow, pathway B). p53 functions as a ‘molecular governor’ of HR. The simplified model shows a single DNA lesion at a replication fork that could represent a carcinogen–DNA adduct, a UV photoproduct or a base damaged by a free radical.

Fig. 6. p53 modulates BLM function during HR and SCE. (A and B) The absence of wild-type p53 and BLM synergistically enhanced the rate of HR in (A) NHF and (B) BS, BS E6, BS A-15 and BS A-15 E6 cell lines. Cells were transfected with either pBHRF alone or with pBHRF with different expression plasmids, as indicated. At 36 h post-transfection, cells were harvested and analyzed by flow cytometry in a FACS Vantage (Becton Dickinson) machine. The fold difference of the GFP/BFP ratio for different sets of transfections is represented. (C) The absence of wild-type p53 further enhances the rate of SCE in BS cells. SCE measurements (mean ± SD) were carried out in BS, BS E6 (pool), BS E6 (clone 1), BS A-15 and BS A-15 E6 (clone 2) fibroblasts, and a diploid control. The P-values were obtained by Student’s t-test.

Fig. 2. BLM, p53 and RAD51 physically interact and co-localize in NHF. (A) Immunoprecipitation (IP) of BLM revealed p53 and RAD51. NHF was either left asynchronous (lane 1) or contact inhibited and treated with HU (lane 2) for 16 h. Lysates (400 µg) were immunoprecipitated with antibodies against BLM (C-18) in the absence (lanes 3 and 4) or presence (lane 5) of the peptide against which the anti-BLM antibody was raised. Input (lanes 1 and 2) indicates 5% of the lysate used for IP. The efficiency of IP was verified with self-antibody (a). Antibodies used for detection of other proteins in BLM IP were (b) anti-p53 (DO-1), (c) anti-phospho-p53 (Ser15) and (d) anti-RAD51 (Ab-1). Asterisks indicates a cross-reactive band. The position of the 46 kDa molecular weight marker is indicated. Lanes 1 and 3, minus HU; lanes 2, 4 and 5, plus HU. (B) IP of p53 revealed RAD51. The same as (A) except that the IP was carried out with antibodies either against p53 (DO-1) (lanes 3 and 4) or against the corresponding IgG (lane 5). Antibodies used for detection of other proteins in p53 IP were (b) anti-phospho-p53 (Ser15) and (c) anti-RAD51 (Ab-1). (C) IP of RAD51 revealed p53 and BLM. The same as (A) except that the IP was carried out with antibodies against RAD51 (Ab-1) (lanes 3 and 4) or against the corresponding IgG (lane 5). Antibodies used for detection of other proteins in RAD51 IP were (b) anti-p53 (DO-1) and (c) anti-BLM (C-18). (D) BLM, p53 and RAD51 co- localized. Contact-inhibited NHF were treated with HU for 16 h. Immunofluorescence was carried out with antibodies against (a) BLM (C-18)/RAD51 (Ab-1) and (b) phospho-p53 (Ser15) 16G8/RAD51 (Ab-1). Quantitation of (a) and (c) is represented in (b) and (d).

Fig. 5. BLM co-localizes and physically interacts with RAD51 even in the absence of p53. (A) BLM co-localized with sites of stalled DNA replication forks in NHF E6. Contact-inhibited NHF E6 cells were treated with HU for 16 h, washed and incubation continued for 10 min in the presence of BrdU. Immunofluorescence were carried out with antibodies against BrdU/BLM (C-18). Quantitation of (a) is represented in (b). (B) BLM and RAD51 interacted in NHF E6. Cells were either asynchronous and left untreated (lanes 1 and 6), or contact inhibited and subsequently treated with HU (lanes 2 and 7) for 16 h. Lysates (400 µg) were immunoprecipitated with antibodies against BLM (C-18, lanes 3–5) or RAD51 (Ab-1, lanes 8 and 9). The peptide against which BLM antibody was raised (lane 5) or the corresponding IgG (lane 10) was used as control for BLM and RAD51 immunoprecipitation (IP), respectively. Input (lanes 1, 2, 6 and 7) indicates 5% of the lysate used for IP. The efficiency of IPs was verified with the respective self-antibodies (a and c). Antibodies against RAD51 (Ab-1) and BLM (C-18) were used for their detection in reciprocal IPs. Lanes 1, 3, 6 and 8, minus HU; lanes 2, 4, 5, 7, 9 and 10, plus HU. (C) BLM co-localized with RAD51. The same as (A) except that immunofluorescence was carried out with antibodies against BLM (C-18)/RAD51 (Ab-1). Quantitation of (a) is represented in (b).

Fig. 3. Ser15-phosphorylated p53 co-localizes with BLM and physically interacts with RAD51 in BS A-15, but not in BS cells. (A) p53 co-localized with BrdU in BS A-15 cells. Contact-inhibited (a) BS or (b) BS A-15 cells were treated with HU for 16 h, washed and incubation continued for 10 min in the presence of BrdU. Immunofluorescence was carried out with antibodies against BrdU/p53 (588). Quantitation of (a) and (b) is represented in (c) and (d). (B) BLM co-localized with BrdU and p53 in BS A-15 cells. The same as (A) except that BS A-15 cells were used. Immunofluorescence was carried out with antibodies against (a) BrdU/BLM (C-18) and (b) BLM (C-18)/p53 (588). Quantitation of (a) and (b) is represented in (c) and (d). (C) Ser15-phosphorylated p53 interacted with RAD51 in BS A-15, but not in BS cells. BS (lanes 1 and 2) or BS A-15 (lanes 6 and 7) cells were either asynchronous and left untreated (lanes 1 and 6) or contact inhibited and subsequently treated with HU (lanes 2 and 7) for 16 h. Lysates (400 µg) were immunoprecipitated with antibodies against p53 (DO-1) (lanes 3, 4, 8 and 9) or against the corresponding IgG (lanes 5 and 10). Input (lanes 1, 2, 6 and 7) indicates 5% of the lysate used for immunoprecipitation (IP). The efficiency of IP was verified with (a) DO-1 and 1801 antibodies. Antibodies used for detection of other proteins in p53 IP were (b) anti-phospho-p53 (Ser15) and (c) anti-RAD51 (Ab-1). The position of the 46 kDa molecular weight marker is indicated in (a) and (b). Lanes 1, 3, 6 and 8, minus HU; lanes 2, 4, 5, 7, 9 and 10, plus HU.

Fig. 4. Wild-type and mutant (Ser15Ala) p53 both co-localize and interact with BLM. (A) BLM accumulated in TR9-7 and TR15-3 cells in S phase. TR9-7 (lanes 1–4) and TR15-3 (lanes 5–8) cells were grown for 16 h in the presence of either 1 µg/ml (lanes 1, 2, 5 and 6) or 50 ng/ml (lanes 3, 4, 7 and 8) tetracycline and in the simultaneous presence or absence of HU. Cell lysates (75 µg) were run on SDS–polyacrylamide gels and western blotted with (a) anti-BLM, (b) anti-p53 (DO-1), (c) anti-p21^WAF1 (Ab-1) and (d) anti-TBP antibodies. The position of the 46 kDa molecular weight marker is indicated. Lanes 1, 3, 5 and 7, minus HU; lanes 2, 4, 6 and 8, plus HU. (B) p53 co-localized with BLM and RAD51 in TR9-7 and TR15-3 cells. The same as (A) except that TR9-7 (a and b) or TR15-3 (c and d) were grown in the presence of 50 ng/ml tetracycline and HU. Immunofluorescence was carried out with antibodies against (a and c) BLM (C-18)/p53 (588) and (b and d) RAD51 (Ab-2)/p53 (588). (C) p53 and RAD51 co-immunoprecipitated with BLM in both TR9-7 and TR15-3 cells. TR9-7 (lanes 1 and 2) or TR15-3 (lanes 6 and 7) cells grown in the presence of 50 ng/ml tetracycline, in the absence (lanes 1 and 6) or presence of HU (lanes 2 and 7) for 16 h. Lysates (400 µg) were immunoprecipitated with antibodies against BLM (C-18) in the absence (lanes 3, 4, 8 and 9) or presence (lanes 5 and 10) of the peptide against which anti-BLM antibody was raised. Input (lanes 1, 2, 6 and 7) indicates 5% of the lysate used for immunoprecipitation (IP). The efficiency of IP was verified with anti-BLM antibody (a). Antibodies used for detection of other proteins in BLM IP were (b) anti-p53 (DO-1) and (c) anti-RAD51 (Ab-1). The position of the 46 kDa molecular weight marker is indicated. Lanes 1, 3, 6 and 8, minus HU; lanes 2, 4, 5, 7, 9 and 10, plus HU.