The replication factory targeting sequence/PCNA‐binding site is required in G 1 to control the phosphorylation status of DNA ligase I

The recruitment of DNA ligase I to replication foci in S phase depends on a replication factory targeting sequence that also mediates the interaction with proliferating cell nuclear antigen (PCNA) in vitro . By exploiting a monoclonal antibody directed at a phospho‐epitope, we demonstrate that Ser66 of DNA ligase I, which is part of a strong CKII consensus site, is phosphorylated in a cell cycle‐dependent manner. After dephosphorylation in early G 1 , the level of Ser66 phosphorylation is minimal in G 1 , increases progressively in S and peaks in G 2 /M phase. The analysis of epitope‐tagged DNA ligase I mutants demonstrates that dephosphorylation of Ser66 requires both the nuclear localization and the PCNA‐binding site of the enzyme. Finally, we show that DNA ligase I and PCNA interact in vivo in G 1 and S phase but not in G 2 /M. We propose that dephosphorylation of Ser66 is part of a novel control mechanism to establish the pre‐replicative form of DNA ligase I.