Published online 23 August 2004
Nucleic Acids Research, Vol. 32 No. 15 © Oxford University Press 2004; all rights reserved
The human Rad9 checkpoint protein stimulates the carbamoyl phosphate synthetase activity of the multifunctional protein CAD
1 Department of Biochemistry and Biophysics, 2 Department of Pharmacology and 3 Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA
* To whom correspondence should be addressed. Tel: +1 919 962 0115; Fax: +1 919 843 8627; Email: Aziz_Sancar{at}med.unc.edu
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
Received July 23, 2004; Revised and Accepted August 6, 2004
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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