The Processing of Holliday Junctions by BLM and WRN Helicases Is Regulated by p53

Abstract

BLM, WRN, and p53 are involved in the homologous DNA recombination pathway. The DNA structure-specific helicases, BLM and WRN, unwind Holliday junctions (HJ), an activity that could suppress inappropriate homologous recombination during DNA replication. Here, we show that purified, recombinant p53 binds to BLM and WRN helicases and attenuates their ability to unwind synthetic HJ in vitro. The p53 248W mutant reduces abilities of both to bind HJ and inhibit helicase activities, whereas the p53 273H mutant loses these abilities. Moreover, full-length p53 and a C-terminal polypeptide (residues 373-383) inhibit the BLM and WRN helicase activities, but phosphorylation at Ser 376 or Ser 378 completely abolishes this inhibition. Following blockage of DNA replication, Ser 15 phospho-p53, BLM, and RAD51 colocalize in nuclear foci at sites likely to contain DNA replication intermediates in cells. Our results are consistent with a novel mechanism for p53-mediated regulation of DNA recombinational repair that involves p53 post-translational modifications and functional protein-protein interactions with BLM and WRN DNA helicases. Bloom and Werner syndromes (BS and WS)1 are autosomal recessive disorders characterized by immune deficiency, cancer predisposition, and chromosomal instability (1). The products of the genes responsible for these disorders, BLM and WRN, are ATP-dependent DNA helicases that exhibit 3Ј to 5Ј polarity. Mutations in the BLM or WRN genes disrupt their helicase activity, which may be important for the phenotypic traits associated with these hereditary diseases (2).Homologous recombination (HR) is required for genetic exchange during meiosis, repair of complex lesions in DNA, and the segregation of chromosomes at cell division. Expression of the BLM or WRN helicases in Saccharomyces cerevisiae containing a mutation in sgs1, a BLM and WRN homolog, suppresses their increased rates of illegitimate recombination and HR (3). BLM and its yeast homologue, Sgs1, functionally interact with topoisomerase III (4), whereas the WRN interaction with DNA polymerase ␦ is apparently required for some aspect of DNA replication and/or repair (5, 6). Recent reports indicate that both of these helicases recognize and disrupt alternative DNA structures, including G-quadruplex DNA and Holliday junctions (HJ) (7-11). HJ arise as intermediates during HR and can occur spontaneously, or during DNA replication and the repair of DNA damage (12). BLM and WRN may promote ATP-dependent translocation of HJ to eliminate DNA recombination intermediates, thereby reducing inappropriate DNA recombination in vivo (10, 11). p53 suppresses genomic instability, particularly in response to DNA damage (13,14). p53 also has been implicated in HR. Evidence of p53 modulation of HR includes the following: (a) overexpression of wild-type p53 (WT p53) can down-regulate the rate of HR between SV40 molecules (15); (b) the rate of HR is increased in p53 mutant cell lines (16 -18); (c) p53 has 3Ј to 5Ј exonuclease and DNA strand transfer activities (19); and...

The mismatch DNA repair heterodimer, hMSH2/6, regulates BLM helicase

“…Statistical analysis showed a significant difference of this BLM binding increase between hMSH2/6 and BSA control group from 20 to 80 nM concentrations (Po0.01). Our previous report showed that 273p53, a recombinant p53 mutant protein that corresponds to a hotspot mutant found in human cancer, did not inhibit the disruption of X-junctions by BLM (Yang et al, 2002). Consistent with these data, 273p53 did not affect BLM binding to X-junctions by ELISA.…”

“…We investigated whether the hMSH2/6 complex modulates BLM activity on HJ by using a radiolabeled HJ mimic, a synthetic X-junction (X-12), as a substrate (McGlynn and Lloyd, 1999). As shown previously (Karow et al, 2000;Yang et al, 2002), purified recombinant BLM disrupted the X-junction (Figure 5a). hMSH2/6 stimulated BLM-mediated disruption in a dose-dependent manner.…”

“…SGS1 is an integral component of the S-phase checkpoint response in yeast and has been directly implicated in S-phase checkpoint activation of signal transduction cascades (Frei and Gasser, 2000). p53 binds to HJ and DNA containing mismatched and bulged bases (Lee et al, 1997;Degtyareva et al, 2001), and regulates BLM to disrupt HJ (Yang et al, 2002). Moreover, p53 physically and functionally interacts with BLM during DNA damage-induced apoptosis .…”

DNA damage tolerance pathway involving DNA polymerase ι and the tumor suppressor p53 regulates DNA replication fork progression

“…To investigate the molecular mechanism underlying p53(WT)-mediated recombination stimulation, we silenced factors implicated in the bypass of blocked replication forks. p53 inhibits the helicase and the branch-migrating activities of Bloom syndrome protein (BLM) and Werner syndrome protein (WRN) helicases, which are involved in the regulation of HR and in the bypass of replication barriers (32,33), whereas RAD51 and breast cancer 2 (BRCA2) are involved in HR-dependent postreplication repair (34,35). Proliferating cell nuclear antigen (PCNA)-associated recombination inhibitor (PARI) associates with DNA damage sites via SUMOylated PCNA and blocks recombination by inhibition of RAD51-DNA filament formation (36).…”

The Werner syndrome protein is required for recruitment of chromatin assembly factor 1 following DNA damage

“…Interestingly, hp150 is not the first common interaction partner of WRN and BLM. BLM and WRN both interact with replication protein A (RPA) (Garcia et al, 2004;Doherty et al, 2005), p53 (Spillare et al, 1999;Wang et al, 2001;Yang et al, 2002), flap endonuclease-1 (FEN-1) (Sharma et al, 2004a, b) and TRF2 Bradshaw et al, 2005). Moreover, BLM and WRN also interact with each other .…”