The replication factory targeting sequence/PCNA- binding site is required in G1 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 prolifer- ating 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 G1, the level of Ser66 phosphorylation is minimal in G1, increases progressively in S and peaks in G2/ 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 G1 and S phase but not in G2/M. We propose that dephosphorylation of Ser66 is part of a novel control mechanism to establish the pre-replicative form of DNA ligase I.cell cycle/CKII/DNA ligase I/phosphorylation/ replication foci