A conserved interaction between the replicative clamp loader and DNA ligase in eukaryotes: implications for Okazaki fragment joining.
The recruitment of DNA ligase I to replication foci and the efficient joining of Okazaki fragments is dependent on the interaction between DNA ligase I and proliferating cell nuclear antigen (PCNA). Although the PCNA sliding clamp tethers DNA ligase I to nicked duplex DNA circles, the interaction does not enhance DNA joining. This suggests that other factors may be involved in the joining of Okazaki fragments. In this study, we describe an association between replication factor C (RFC), the clamp loader, and DNA ligase I in human cell extracts. Subsequently, we demonstrate that there is a direct physical interaction between these proteins that involves both the N- and C-terminal domains of DNA ligase I, the N terminus of the large RFC subunit p140, and the p36 and p38 subunits of RFC. Although RFC inhibited DNA joining by DNA ligase I, the addition of PCNA alleviated inhibition by RFC. Notably, the effect of PCNA on ligation was dependent on the PCNA-binding site of DNA ligase I. Together, these results provide a molecular explanation for the key in vivo role of the DNA ligase I/PCNA interaction and suggest that the joining of Okazaki fragments is coordinated by pairwise interactions among RFC, PCNA, and DNA ligase I.
Levin
David S
DS
Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center, San Antonio, TX 78245, USA.
Vijayakumar
Sangeetha
S
Liu
Xiuping
X
Bermudez
Vladimir P
VP
Hurwitz
Jerard
J
Tomkinson
Alan E
AE
D001665:Binding Sites; D001710:Biotin / Q000494:pharmacology; D002845:Chromatography; D004247:DNA / Q000737:chemistry / Q000502:physiology*; D011088:DNA Ligases / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000737:chemistry; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D018809:Proliferating Cell Nuclear Antigen / Q000502:physiology*; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D051818:Replication Protein C; D013997:Time Factors; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C022262:Okazaki fragments; D018809:Proliferating Cell Nuclear Antigen; D001710:Biotin; D004247:DNA; D005982:Glutathione Transferase; D051818:Replication Protein C; D011088:DNA Ligases