A conserved interaction between the replicative clamp loader and DNA ligase in eukaryotes: implications for Okazaki...

Area of Science:

• Molecular Biology
• Biochemistry
• Cellular Processes

Background:

• DNA ligase I and proliferating cell nuclear antigen (PCNA) interaction is crucial for Okazaki fragment joining during DNA replication.
• While PCNA tethers DNA ligase I to DNA, this interaction alone does not enhance ligation efficiency, suggesting additional regulatory factors.
• The precise molecular mechanisms coordinating Okazaki fragment joining remain incompletely understood.

Purpose of the Study:

• To investigate potential novel interactions involving DNA ligase I in Okazaki fragment joining.
• To elucidate the role of replication factor C (RFC) in the DNA ligase I-mediated ligation process.
• To understand how RFC and PCNA cooperate with DNA ligase I during DNA replication.

Main Methods:

• Co-immunoprecipitation assays using human cell extracts to detect protein-protein interactions.
• In vitro biochemical assays to assess the effects of RFC and PCNA on DNA ligase I activity.
• Mapping of protein interaction domains within DNA ligase I, RFC subunits (p140, p36, p38), and PCNA.

Main Results:

• A physical association between DNA ligase I and replication factor C (RFC) was identified in human cell extracts.
• Direct interactions were confirmed between DNA ligase I (N- and C-terminal domains) and RFC subunits (p140, p36, p38).
• RFC inhibited DNA ligase I activity, but this inhibition was alleviated by PCNA in a manner dependent on the PCNA-binding site of DNA ligase I.

Conclusions:

• The study reveals a novel interaction between DNA ligase I and RFC, a clamp loader complex.
• These findings provide a molecular basis for the essential in vivo role of the DNA ligase I/PCNA interaction in replication.
• Okazaki fragment joining is likely a coordinated process involving dynamic, pairwise interactions among RFC, PCNA, and DNA ligase I.