Requirement for ATP by the DNA damage checkpoint clamp loader.
The DNA damage clamp loader replication factor C (RFC-Rad24) consists of the Rad24 protein and the four small Rfc2-5 subunits of RFC. This complex loads the heterotrimeric DNA damage clamp consisting of Rad17, Mec3, and Ddc1 (Rad17/3/1) onto partial duplex DNA in an ATP-dependent manner. Interactions between the clamp loader and the clamp have been proposed to mirror those of the replication clamp loader RFC and the sliding clamp proliferating cell nuclear antigen (PCNA). In that system, three ATP molecules bound to the Rfc2, Rfc3, and Rfc4 subunits are necessary and sufficient for efficient loading of PCNA, whereas ATP binding to Rfc1 is not required. In contrast, in this study, we show that mutant RFC-Rad24 with a rad24-K115E mutation in the ATP-binding domain of Rad24 shows defects in the ATPase of the complex and is defective for interaction with Rad17/3/1 and for loading of the checkpoint clamp. A similar defect was measured with a mutant RFC-Rad24 clamp loader carrying a rfc4K55R ATP-binding mutation, whereas the rfc4K55E clamp loader showed partial loading activity, in agreement with genetic studies of these mutants. These studies show that ATP utilization by the checkpoint clamp/clamp loader system is effectively different from that by the structurally analogous replication system.
Majka
Jerzy
J
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
Chung
Brian Y
BY
Burgers
Peter M J
PM
D048868:Adaptor Proteins, Signal Transducing; D000251:Adenosine Triphosphatases / Q000032:analysis; D000255:Adenosine Triphosphate / Q000378:metabolism*; D018797:Cell Cycle Proteins; D004249:DNA Damage / Q000502:physiology*; D004261:DNA Replication / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009687:Nuclear Proteins / Q000378:metabolism*; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C413163:MDC1 protein, human; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases