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The human Rad9 checkpoint protein stimulates the carbamoyl phosphate synthetase activity of the multifunctional protein CAD.

Authors
  • Lindsey-Boltz, Laura A
  • Wauson, Eric M
  • Graves, Lee M
  • Sancar, Aziz
Type
Published Article
Journal
Nucleic Acids Research
Publisher
Oxford University Press
Publication Date
Jan 01, 2004
Volume
32
Issue
15
Pages
4524–4530
Identifiers
PMID: 15326225
Source
Medline
License
Unknown

Abstract

The human Rad9 checkpoint protein is a subunit of the heterotrimeric Rad9-Rad1-Hus1 (9-1-1) complex that plays a role as a damage sensor in the DNA damage checkpoint response. Rad9 has been found to interact with several other proteins outside the context of the 9-1-1 complex with no obvious checkpoint functions. During our studies on the 9-1-1 complex, we found that Rad9 immunoprecipitates contained a 240 kDa protein that was identified as carbamoyl phosphate synthetase/aspartate transcarbamoylase/dihydroorotase (CAD), a multienzymatic protein required for the de novo synthesis of pyrimidine nucleotides and cell growth. Further investigations revealed that only free Rad9, but not Rad9 within the 9-1-1 complex, bound to CAD. The rate-limiting step in de novo pyrimidine nucleotide synthesis is catalyzed by the carbamoyl phosphate synthetase II (CPSase) domain of CAD. We find that Rad9 binds to the CPSase domain, and, moreover, this binding results in a 2-fold stimulation of the CPSase activity of CAD. Similar results were also obtained with an N-terminal Rad9 fragment. These findings suggest that Rad9 may play a role in ribonucleotide biosynthesis.

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