Structures of the human Rad17-replication factor C and checkpoint Rad 9-1-1 complexes visualized by glycerol spray/low voltage microscopy.
Human checkpoint Rad proteins are thought to function as damage sensors in the DNA damage checkpoint response pathway. The checkpoint proteins hRad9, hHus1, and hRad1 have limited homology to the replication processivity factor proliferating cell nuclear antigen (PCNA), and hRad17 has homology to replication factor C (RFC). Such observations have led to the proposal that these checkpoint Rad proteins may function similarly to their replication counterparts during checkpoint control. We purified two complexes formed by the checkpoint Rad proteins and investigated their structures using an electron microscopic preparative method in which the complexes are sprayed from a glycerol solution onto very thin carbon foils, decorated in vacuo with tungsten, and imaged at low voltage. We found that the hRad9, hHus1, and hRad1 proteins make a trimeric ring structure (checkpoint 9-1-1 complex) reminiscent of the PCNA ring. Similarly we found that hRad17 makes a heteropentameric complex with the four RFC small subunits (hRad17-RFC) with a deep groove or cleft and is similar to the RFC clamp loader. Therefore, our results demonstrate structural similarity between the checkpoint Rad complexes and the PCNA and RFC replication factors and thus provide further support for models proposing analogous functions for these complexes.
Griffith
Jack D
JD
Department of Biochemistry and Biophysics and Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA.
Lindsey-Boltz
Laura A
LA
Sancar
Aziz
A
D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000648:ultrastructure; D005990:Glycerol; D006801:Humans; D008854:Microscopy, Electron; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000648:ultrastructure; D011994:Recombinant Proteins / Q000737:chemistry / Q000648:ultrastructure; D051818:Replication Protein C; D014162:Transfection; D018631:ras Proteins
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D018809:Proliferating Cell Nuclear Antigen; C084355:RRAD protein, human; D011994:Recombinant Proteins; D051818:Replication Protein C; D018631:ras Proteins; D005990:Glycerol