Replication protein A-mediated recruitment and activation of Rad17 complexes.
The human Rad17-Rfc2-5 and Rad9-Rad1-Hus1 complexes play crucial roles in the activation of the ATR-mediated DNA damage and DNA replication stress response pathways. In response to DNA damage, Rad9 is recruited to chromatin in a Rad17-dependent manner in human cells. However, the DNA structures recognized by the Rad17-Rfc2-5 complex during the damage response have not been defined. Here, we show that replication protein A (RPA) stimulates the binding of the Rad17-Rfc2-5 complex to single-stranded DNA (ssDNA), primed ssDNA, and a gapped DNA structure. Furthermore, RPA facilitates the recruitment of the Rad9-Rad1-Hus1 complex by the Rad17-Rfc2-5 complex to primed and gapped DNA structures in vitro. These findings suggest that RPA-coated ssDNA is an important part of the structures recognized by the Rad17-Rfc2-5 complex. Unlike replication factor C (RFC), which uses the 3' primer/template junction to recruit proliferating cell nuclear antigen (PCNA), the Rad17-Rfc2-5 complex can use both the 5' and the 3' primer/template junctions to recruit the Rad9-Rad1-Hus1 complex, and it shows a preference for gapped DNA structures. These results explain how the Rad17-Rfc2-5 complex senses DNA damage and DNA replication stress to initiate checkpoint signaling.
Zou
Lee
L
Brigham and Women's Hospital, Howard Hughes Medical Institute, Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
Liu
Dou
D
Elledge
Stephen J
SJ
D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008954:Models, Biological; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051716:Replication Protein A; D051818:Replication Protein C; D012441:Saccharomyces cerevisiae / Q000235:genetics
D018797:Cell Cycle Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; C497561:RFC2 protein, human; C497562:RFC3 protein, human; C497167:RPA1 protein, human; C113575:Rad17 protein, human; D011994:Recombinant Proteins; D051716:Replication Protein A; C069930:rad9 protein; D051818:Replication Protein C