Phosphorylation-dependent interactions of BLM and 53BP1 are required for their anti-recombinogenic roles during homologous recombination.
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.
Tripathi
Vivek
V
National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
Kaur
Sarabpreet
S
Sengupta
Sagar
S
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D015153:Blotting, Western; D002461:Cell Line, Transformed; D004265:DNA Helicases / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D051135:Rad51 Recombinase / Q000378:metabolism; D053484:RecQ Helicases; D011995:Recombination, Genetic; D000071857:Tumor Suppressor p53-Binding Protein 1
D047908:Intracellular Signaling Peptides and Proteins; C498301:TP53BP1 protein, human; D000071857:Tumor Suppressor p53-Binding Protein 1; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D000251:Adenosine Triphosphatases; C097151:Bloom syndrome protein; D004265:DNA Helicases; D053484:RecQ Helicases