Phosphorylation-dependent interactions of BLM and 53BP1 are required for their anti-recombinogenic roles during homologous recombination

Abstract

Mutations in bloom helicase protein (BLM) helicase cause Bloom syndrome, characterized by predisposition to almost all forms of cancer. We have demonstrated previously that endogenous BLM, signal transducer 53BP1 and RAD51 are present in a complex during replication stress. Using full-length recombinant proteins, we now provide evidence that these proteins physically interact. BLM interacts with checkpoint kinase (Chk) 1 via the kinetochore-binding domain (KBD). Wild-type (WT) Chk1 phosphorylates 53BP1 in the KBD, both in vitro and in vivo during replication stress. Chk1-mediated phosphorylation of 53BP1 enhances its binding to BLM and is required for the accumulation of 53BP1 at the site of stalled replication. 53BP1, in turn, binds to the N-terminal domain of BLM. Ataxia telangiectasia and Rad3 related (ATR)-mediated phosphorylation of BLM at Thr99 is critical for its interaction and subsequent co-localization with 53BP1. WT BLM enhances the interaction and co-localization between 53BP1 and RAD51 during replication arrest. Interactions between the three proteins have functional consequences. Non-binding or phosphorylation-deficient mutants of BLM and 53BP1 fail to demonstrate the anti-recombinogenic property of the WT counterparts. Consequently, these mutants cause elevation of endogenous RAD51 foci formation. These results provide evidence that the phosphorylation-mediated interactions between BLM, 53BP1 and RAD51 are required for their regulatory roles during homologous recombination.

BLM helicase stimulates the ATPase and chromatin-remodeling activities of RAD54

“…Using an in vitro GST pulldown assay, we found that full-length BLM and RAD54 directly interacted. Using purified BLM fragments (Tripathi et al, 2008), we further demonstrated that the N-terminal 212 amino acids of BLM mediated this interaction (Fig. 2B).…”

“…Hence, the question arises: how does BLM switch its function from a prorecombinogenic to an anti-recombinogenic protein? We have recently demonstrated that lack of ATR-mediated BLM phosphorylation on Thr99 prevents the helicase from disrupting RAD51 polymerization (Tripathi et al, 2008). Additional cellular and molecular event(s) including additional post-translational modification(s) on BLM and/or interaction with other antirecombinogenic proteins, such as 53BP1 and p53 (Sengupta et al, 2003;Tripathi et al, 2007) might also be required during this transition process.…”

“…Using genetic and cell biology approaches, we recently provided in vivo evidence that BLM can abrogate endogenous RAD51 foci formation and disrupt RAD51 polymerization (Tripathi et al, 2007). Phosphorylation of BLM at Thr99 by ATR is required for the disruption of RAD51 filaments (Tripathi et al, 2008). In vitro biochemical evidence confirmed that BLM can dislodge RAD51 from ssDNA in an ATPase-dependent manner (Bugreev et al, 2007).…”

Enhancement of c-Myc degradation by Bloom (BLM) helicase leads to delayed tumor initiation

“…Recombinants and siRNA pGEX4T-1 BLM (1-1417) (Srivastava et al, 2009), pGEX4T-1 BLM (1-212), pcDNA3 Flag BLM (gift from Ian Hickson), EGFP-C1 BLM (gift from Nathan Ellis), pGEX4T-1 BLM (191-660), pGEX4T-1 BLM (621-1041), pGEX4T-1 BLM (1001-1417) (Tripathi et al, 2008). pSG5-Jun (gift from Bohdan Wasylyk).…”

“…BS patients exhibit defects in DNA replication and homologous recombination events, manifested by an increased frequency of sister chromatid exchanges (SCEs) (Chaganti et al, 1974). BLM regulates homologous recombination by a number of mechanisms, such as disruption of RAD51 nucleofilaments (Bugreev et al, 2007;Tripathi et al, 2007), prevention of chromosome breakage (Russell et al, 2011), recruitment of p53 to carry out its own functions during homologous recombination (Sengupta et al, 2003) and enhancement of the interactions between 53BP1 and RAD51 (Tripathi et al, 2008;Tripathi et al, 2007). BLM is involved in the recognition of the DNA damage by a K63-linked ubiquitindependent mechanism (Tikoo et al, 2013), transmission of the damage signal to the repair proteins and in repair of the DNA damage during the effector phase (Tikoo and Sengupta, 2010).…”