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A second proliferating cell nuclear antigen loader complex, Ctf18-replication factor C, stimulates DNA polymerase eta

Yasushi Shiomi¹, Chikahide Masutani, Fumio Hanaoka

¹Department of Biology, School of Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.

The Journal of Biological Chemistry

|June 5, 2007

Summary

Ctf18-RFC, a novel proliferating cell nuclear antigen (PCNA) loader, directly interacts with and stimulates DNA polymerase eta. This interaction helps DNA replication forks navigate challenging DNA structures, including damaged DNA and protein complexes.

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Area of Science:

Molecular Biology
DNA Replication
Chromosomal Cohesion

Background:

Replication factor C (RFC) is essential for loading the proliferating cell nuclear antigen (PCNA) clamp onto DNA.
Ctf18-RFC is a distinct RFC complex containing chromosome cohesion factors, functioning as an alternative PCNA loader.
Understanding Ctf18-RFC's function requires identifying its specific DNA polymerase targets.

Purpose of the Study:

To identify DNA polymerases that are specifically stimulated by the Ctf18-RFC complex.
To investigate the functional interaction between Ctf18-RFC and its potential DNA polymerase targets.
To elucidate the role of Ctf18-RFC in facilitating DNA replication through challenging DNA structures.

Main Methods:

Assaying human cell extracts for Ctf18-RFC-dependent DNA polymerase activity.
Purification of the active complex using chromatography.
Liquid chromatography/tandem mass spectrometry (LC/MS/MS) for protein identification.
Biochemical assays with purified recombinant proteins.
Co-precipitation assays to confirm physical interactions.

Main Results:

A novel DNA polymerase activity stimulated by Ctf18-RFC was identified and purified.
Liquid chromatography/tandem mass spectrometry identified DNA polymerases eta and lambda.
Biochemical experiments confirmed that DNA polymerase eta, not lambda, was responsible for the activity.
Ctf18-RFC alone stimulated polymerase eta, with synergistic stimulation upon PCNA addition.
Physical interaction between Ctf18-RFC and polymerase eta was demonstrated via co-precipitation.

Conclusions:

Ctf18-RFC directly interacts with and stimulates DNA polymerase eta activity.
This interaction is enhanced by PCNA, suggesting a cooperative mechanism.
The Ctf18-RFC-polymerase eta interaction likely facilitates replication fork progression through difficult DNA templates.
This pathway is crucial for maintaining genome stability by overcoming DNA damage and protein-DNA obstacles during replication.