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.
Rossi
R
R
Istituto di Genetica Biochimica ed Evoluzionistica, CNR, Via Abbiategrasso 207, 27100 Pavia, Italy.
Villa
A
A
Negri
C
C
Scovassi
I
I
Ciarrocchi
G
G
Biamonti
G
G
Montecucco
A
A
D016475:3T3 Cells; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D019556:COS Cells; D002460:Cell Line; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D004261:DNA Replication / Q000502:physiology*; D000939:Epitopes / Q000737:chemistry; D016193:G1 Phase / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D010766:Phosphorylation; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D016196:S Phase / Q000502:physiology; D012694:Serine / Q000737:chemistry
D000911:Antibodies, Monoclonal; D000939:Epitopes; C000607892:LIG1 protein, human; D018809:Proliferating Cell Nuclear Antigen; D012694:Serine; D011088:DNA Ligases; D000072481:DNA Ligase ATP