Replication protein A directs loading of the DNA damage checkpoint clamp to 5'-DNA junctions.
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.
Majka
Jerzy
J
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Binz
Sara K
SK
Wold
Marc S
MS
Burgers
Peter M J
PM
D000251:Adenosine Triphosphatases / Q000502:physiology; D018797:Cell Cycle Proteins / Q000502:physiology; D004249:DNA Damage; D004265:DNA Helicases / Q000502:physiology; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins / Q000502:physiology; D004720:Endonucleases / Q000502:physiology; D047908:Intracellular Signaling Peptides and Proteins / Q000502:physiology; D009687:Nuclear Proteins / Q000502:physiology; D018809:Proliferating Cell Nuclear Antigen / Q000502:physiology; D051716:Replication Protein A / Q000502:physiology*; D051818:Replication Protein C / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000502:physiology; D013379:Substrate Specificity
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; C501395:RAD17 protein, S cerevisiae; C088385:RAD24 protein, S cerevisiae; C084903:RFC1 protein, S cerevisiae; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; D004720:Endonucleases; C069266:RAD1 protein, S cerevisiae; D000251:Adenosine Triphosphatases; C061897:Rad3 protein, S cerevisiae; D004265:DNA Helicases; D051818:Replication Protein C; D045643:DNA Repair Enzymes