A second DNA binding site on RFC facilitates clamp loading at gapped or nicked DNA

Abstract
Data availability
Article and author information
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Abstract

Clamp loaders place circular sliding clamp proteins onto DNA so that clamp-binding partner proteins can synthesize, scan, and repair the genome. DNA with nicks or small single-stranded gaps are common clamp-loading targets in DNA repair, yet these substrates would be sterically blocked given the known mechanism for binding of primer-template DNA. Here, we report the discovery of a second DNA binding site in the yeast clamp loader Replication Factor C (RFC) that aids in binding to nicked or gapped DNA. This DNA binding site is on the external surface and is only accessible in the open conformation of RFC. Initial DNA binding at this site thus provides access to the primary DNA binding site in the central chamber. Furthermore, we identify that this site can partially unwind DNA to create an extended single-stranded gap for DNA binding in RFC's central chamber and subsequent ATPase activation. Finally, we show that deletion of the BRCT domain, a major component of the external DNA binding site, results in defective yeast growth in the presence of DNA damage where nicked or gapped DNA intermediates occur. We propose that RFC’s external DNA binding site acts to enhance DNA binding and clamp loading, particularly at DNA architectures typically found in DNA repair.

Data availability

The reported cryo-EM map and atomic coordinates have been deposited in the Electron Microscopy Data Bank (entry numbers EMD-26280EMD-26298EMD-26302EMD-26297) and the Protein Data Bank (ID codes 7U1A, 7U1P, 7U19).

The following data sets were generated

1. Liu
2. Gaubitz
3. Pajak
4. Kelch
(2022) RFC bound to PCNA and two primer/template DNA molecules
EMD-26280.

https://www.ebi.ac.uk/emdb/
1. Liu
2. Gaubitz
3. Pajak
4. Kelch
(2022) RFC:PCNA bound to dsDNA with a ssDNA gap of six nucleotides
PDB 7U1A, EMD-26298.

https://doi.org/10.2210/pdb7U1A/pdb
1. Liu
2. Gaubitz
3. Pajak
4. Kelch
(2022) RFC:PCNA bound to DNA with a ssDNA gap of five nucleotides
PDB 7U1P, EMD-26302.

https://doi.org/10.2210/pdb7U1P/pdb
1. Liu
2. Gaubitz
3. Pajak
4. Kelch
(2022) RFC:PCNA bound to nicked DNA
PDB 7U19, EMD-26297.

https://doi.org/10.2210/pdb7U19/pdb

Article and author information

Author details

Xingchen Liu

Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Medical School, Worcester, United States

Competing interests
The authors declare that no competing interests exist.
Christl Gaubitz

Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Medical School, Worcester, United States

For correspondence
cgaubitz@sund.ku.dk

Competing interests
The authors declare that no competing interests exist.
Joshua Pajak

Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Medical School, Worcester, United States

Competing interests
The authors declare that no competing interests exist.
Brian A Kelch

Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Medical School, Worcester, United States

For correspondence
brian.kelch@umassmed.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-1369-6989

Funding

NIGMS (1R01GM127776-01A1)

Brian A Kelch

Swiss National Science Foundation (177859)

Christl Gaubitz

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.