DNA ligase I and proliferating cell nuclear antigen form a functional complex

DNA ligase I is responsible for joining Okazaki fragments during DNA replic ation. An additional proposed role for DNA ligase I is sealing nicks genera ted during excision repair, Previous studies have shown that there is a phy sical interaction between DNA ligase I and proliferating cell nuclear antig en (PCNA), another important component of DNA replication and repair. The r esults shown here indicate that human PCNA enhances the reaction rate of hu man 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 assay s indicate that PCNA improves the binding of DNA ligase I to the ligation s ite. Increasing the DNA ligase I concentration leads to a reduction in PCNA stimulation, consistent with PCNA-directed improvement of DNA ligase I bin ding to its DNA substrate. Two experiments show that PCNA is required to en circle duplex DNA to enhance DNA ligase I activity. Biotin-streptavidin con jugations at the ends of a linear substrate inhibit PCNA stimulation. PCNA cannot enhance ligation on a circular substrate without the addition of rep lication factor C, which is the protein responsible for loading PCNA onto d uplex DNA. These results show that PCNA is responsible for the stable assoc iation of DNA ligase I to nicked duplex DNA.