The BRCT domain and the specific loop 1 of human Polμ are targets of Cdk2/cyclin A phosphorylation

• Cdk2/cyclin A complex phosphorylates human Polμ in vitro.
• Ser12, Thr21 (BRCT domain) and Ser372 (loop1) are the Cdk target residues.
• Phospho-mimicking S372E impaired terminal transferase and end-joining activities.
• Polμ gap-filling activity was not affected by S372E mutation.
• Polμ error-prone activities might be cell cycle regulated by phosphorylation.

Human family X polymerases contribute both to genomic stability and variability through their specialized functions in DNA repair. Polμ participates in the repair of spontaneous double strand breaks (DSB) by non homologous end-joining (NHEJ), and also in the V(D)J recombination process after programmed DSBs. Polμ plays this dual role due to its template-dependent and terminal transferase (template-independent) polymerization activities. In this study we evaluated if Polμ could be regulated by Cdk phosphorylation along the cell cycle. In vitro kinase assays showed that the S phase-associated Cdk2/cyclin A complex was able to phosphorylate Polμ. We identified Ser12, Thr21 (located in the BRCT domain) and Ser372 (located in loop1) as the target residues. Mutation of these residues to alanine indicated that Ser372 is the main phosphorylation site. Mobilization of loop1, which mediates DNA end micro-synapsis, is crucial both for terminal transferase and NHEJ. Interestingly, the phospho-mimicking S372E mutation specifically impaired these activities. Our evidences suggest that Polμ could be regulated in vivo by phosphorylation of the BRCT domain (Ser12/Thr21) and of Ser372, affecting the function of loop1. Consequently, Polμ’s most distinctive activities would be turned off at specific cell-cycle phases (S and G2), when these promiscuous functions might be harmful to the cell.