Phosphorylation of human DNA ligase I regulates its interaction with replication factor C and its participation in DNA replication and DNA repair.
Human DNA ligase I (hLigI) participates in DNA replication and excision repair via an interaction with proliferating cell nuclear antigen (PCNA), a DNA sliding clamp. In addition, hLigI interacts with and is inhibited by replication factor C (RFC), the clamp loader complex that loads PCNA onto DNA. Here we show that a mutant version of hLigI, which mimics the hyperphosphorylated M-phase form of hLigI, does not interact with and is not inhibited by RFC, demonstrating that inhibition of ligation is dependent upon the interaction between hLigI and RFC. To examine the biological relevance of hLigI phosphorylation, we isolated derivatives of the hLigI-deficient cell line 46BR.1G1 that stably express mutant versions of hLigI in which four serine residues phosphorylated in vivo were replaced with either alanine or aspartic acid. The cell lines expressing the phosphorylation site mutants of hLigI exhibited a dramatic reduction in proliferation and DNA synthesis and were also hypersensitive to DNA damage. The dominant-negative effects of the hLigI phosphomutants on replication and repair are due to the activation of cellular senescence, presumably because of DNA damage arising from replication abnormalities. Thus, appropriate phosphorylation of hLigI is critical for its participation in DNA replication and repair.
Vijayakumar
Sangeetha
S
Department of Radiation Oncology, University of Maryland School of Medicine, Baltimore, MD 21201-1509, USA.
Dziegielewska
Barbara
B
Levin
David S
DS
Song
Wei
W
Yin
Jinhu
J
Yang
Austin
A
Matsumoto
Yoshihiro
Y
Bermudez
Vladimir P
VP
Hurwitz
Jerard
J
Tomkinson
Alan E
AE
D002460:Cell Line; D049109:Cell Proliferation; D016922:Cellular Senescence; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D004261:DNA Replication; D006801:Humans; D050505:Mutant Proteins; D010766:Phosphorylation; D051818:Replication Protein C / Q000378:metabolism* / Q000502:physiology
C000607892:LIG1 protein, human; D050505:Mutant Proteins; D051818:Replication Protein C; D011088:DNA Ligases; D000072481:DNA Ligase ATP