DNA ligase I and proliferating cell nuclear antigen form a functional complex.
DNA ligase I is responsible for joining Okazaki fragments during DNA replication. An additional proposed role for DNA ligase I is sealing nicks generated during excision repair. Previous studies have shown that there is a physical interaction between DNA ligase I and proliferating cell nuclear antigen (PCNA), another important component of DNA replication and repair. The results shown here indicate that human PCNA enhances the reaction rate of human DNA ligase I up to 5-fold. The stimulation is specific to DNA ligase I because T4 DNA ligase is not affected. Electrophoretic mobility shift assays indicate that PCNA improves the binding of DNA ligase I to the ligation site. Increasing the DNA ligase I concentration leads to a reduction in PCNA stimulation, consistent with PCNA-directed improvement of DNA ligase I binding to its DNA substrate. Two experiments show that PCNA is required to encircle duplex DNA to enhance DNA ligase I activity. Biotin-streptavidin conjugations at the ends of a linear substrate inhibit PCNA stimulation. PCNA cannot enhance ligation on a circular substrate without the addition of replication factor C, which is the protein responsible for loading PCNA onto duplex DNA. These results show that PCNA is responsible for the stable association of DNA ligase I to nicked duplex DNA.
Tom
S
S
Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave., Rochester, NY 14642, USA.
Henricksen
L A
LA
Park
M S
MS
Bambara
R A
RA
D047390:Casein Kinase II; D004247:DNA / Q000378:metabolism; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D010766:Phosphorylation; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism
C000607892:LIG1 protein, human; D046911:Macromolecular Substances; C022262:Okazaki fragments; D018809:Proliferating Cell Nuclear Antigen; D004247:DNA; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D011088:DNA Ligases; D000072481:DNA Ligase ATP