title: Phosphorylation of serine 51 regulates the interaction of human DNA ligase I with replication factor C and its participation in DNA replication and repair.
abstract: Human DNA ligase I (hLigI) joins Okazaki fragments during DNA replication and completes excision repair via interactions with proliferating cell nuclear antigen and replication factor C (RFC). Unlike proliferating cell nuclear antigen, the interaction with RFC is regulated by hLigI phosphorylation. To identity of the site(s) involved in this regulation, we analyzed phosphorylated hLigI purified from insect cells by mass spectrometry. These results suggested that serine 51 phosphorylation negatively regulates the interaction with RFC. Therefore, we constructed versions of hLigI in which serine 51 was replaced with either alanine (hLigI51A) to prevent phosphorylation or aspartic acid (hLigI51D) to mimic phosphorylation. hLigI51D but not hLigI51A was defective in binding to purified RFC and in associating with RFC in cell extracts. Although DNA synthesis and proliferation of hLigI-deficient cells expressing either hLig51A or hLig51 was reduced compared with cells expressing wild-type hLigI, cellular senescence was only observed in the cells expressing hLigI51D. Notably, these cells had increased levels of spontaneous DNA damage and phosphorylated CHK2. In addition, although expression of hLigI51A complemented the sensitivity of hLigI-deficient cells to a poly (ADP-ribose polymerase (PARP) inhibitor, expression of hLig151D did not, presumably because these cells are more dependent upon PARP-dependent repair pathways to repair the damage resulting from the abnormal DNA replication. Finally, neither expression of hLigI51D nor hLigI51A fully complemented the sensitivity of hLigI-deficient cells to DNA alkylation. Thus, phosphorylation of serine 51 on hLigI plays a critical role in regulating the interaction between hLigI and RFC, which is required for efficient DNA replication and repair.
authors.affiliation: Department of Internal Medicine and University of New Mexico Cancer Center, University of New Mexico, Albuquerque, New Mexico 87131, USA.
authors.forename: Zhimin
authors.initials: Z
authors.lastname: Peng
authors.forename: Zhongping
authors.initials: Z
authors.lastname: Liao
authors.forename: Barbara
authors.initials: B
authors.lastname: Dziegielewska
authors.forename: Yoshi
authors.initials: Y
authors.lastname: Matsumoto
authors.forename: Stefani
authors.initials: S
authors.lastname: Thomas
authors.forename: Yunhu
authors.initials: Y
authors.lastname: Wan
authors.forename: Austin
authors.initials: A
authors.lastname: Yang
authors.forename: Alan E
authors.initials: AE
authors.lastname: Tomkinson
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation; D048430:Cell Shape; D016922:Cellular Senescence; D004249:DNA Damage; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000737:chemistry / Q000378:metabolism*; D004260:DNA Repair; D004261:DNA Replication; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D010766:Phosphorylation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D051818:Replication Protein C / Q000737:chemistry / Q000378:metabolism*; D012694:Serine / Q000378:metabolism
chemical_list: C000607892:LIG1 protein, human; D010446:Peptide Fragments; C081632:RFC1 protein, human; D012694:Serine; D051818:Replication Protein C; D011088:DNA Ligases; D000072481:DNA Ligase ATP