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 mo noclonal antibody directed at a phospho-epitope, we demonstrate that Ser66 of DNA ligase I, which is part of a strong CKII consensus site, is phosphor ylated in a cell cycle-dependent manner. After dephosphorylation in early G (1), the level of Ser66 phosphorylation is minimal in G(1), increases progr essively in S and peaks in G(2)/M phase. The analysis of epitope-tagged DNA ligase I mutants demonstrates that dephosphorylation of Ser66 requires bot h 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 b ut not in G(2)/M, We propose that dephosphorylation of Ser66 is part of a n ovel control mechanism to establish the pre-replicative form of DNA ligase I.