Replication protein A directs loading of the DNA damage checkpoint clamp to 5'-DNA junctions

Jerzy Majka; Sara K Binz; Marc S Wold; Peter M J Burgers

doi:10.1074/jbc.M605176200

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Replication protein A directs loading of the DNA damage checkpoint clamp to 5'-DNA junctions

Journal article

Open access

Peer reviewed

Replication protein A directs loading of the DNA damage checkpoint clamp to 5'-DNA junctions

Jerzy Majka, Sara K Binz, Marc S Wold and Peter M J Burgers

The Journal of biological chemistry, Vol.281(38), pp.27855-27861

09/22/2006

DOI: 10.1074/jbc.M605176200

PMID: 16864589

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Abstract

The heterotrimeric checkpoint clamp comprises the Saccharomyces cerevisiae Rad17, Mec3, and Ddc1 subunits (Rad17/3/1, the 9-1-1 complex in humans). This DNA damage response factor is loaded onto DNA by the Rad24-RFC (replication factor C-like complex with Rad24) clamp loader and ATP. Although Rad24-RFC alone does not bind to naked partial double-stranded DNA, coating of the single strand with single-stranded DNA-binding protein RPA (replication protein A) causes binding of Rad24-RFC via interactions with RPA. However, RPA-mediated binding is abrogated when the DNA is coated with RPA containing a rpa1-K45E (rfa1-t11) mutation. These properties allowed us to determine the role of RPA in clamp-loading specificity. The Rad17/3/1 clamp is loaded with comparable efficiency onto naked primer/template DNA with either a 3'-junction or a 5'-junction. Remarkably, when the DNA was coated with RPA, loading of Rad17/3/1 at 3'-junctions was completely inhibited, thereby providing specificity to loading at 5'-junctions. However, Rad17/3/1 loaded at 5'-junctions can slide across double-stranded DNA to nearby 3'-junctions and thereby affect the activity of proteins that act at 3'-termini. These studies show a unique specificity of the checkpoint loader for 5'-junctions of RPA-coated DNA. The implications of this specificity for checkpoint function are discussed.

DNA-Binding Proteins - physiology

Replication Protein A - physiology

Substrate Specificity

DNA Repair Enzymes

Proliferating Cell Nuclear Antigen - physiology

Nuclear Proteins - physiology

Saccharomyces cerevisiae Proteins - physiology

DNA Damage

Intracellular Signaling Peptides and Proteins - physiology

Adenosine Triphosphatases - physiology

Cell Cycle Proteins - physiology

Endonucleases - physiology

Replication Protein C - physiology

DNA Helicases - physiology

Details

Title: Subtitle: Replication protein A directs loading of the DNA damage checkpoint clamp to 5'-DNA junctions
Creators: Jerzy Majka - Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA
Sara K Binz
Marc S Wold
Peter M J Burgers
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.281(38), pp.27855-27861
DOI: 10.1074/jbc.M605176200
PMID: 16864589
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Publisher: United States
Grant note: GM44721 / NIGMS NIH HHS GM32431 / NIGMS NIH HHS R01 GM032431 / NIGMS NIH HHS
Language: English
Date published: 09/22/2006
Academic Unit: Radiation Oncology; Biochemistry and Molecular Biology
Record Identifier: 9984024415102771

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