A novel protein activity mediates DNA binding of an ATR-ATRIP complex.
The function of the ATR (ataxia-telangiectasia mutated and Rad3-related)-ATRIP (ATR-interacting protein) protein kinase complex is central to the cellular response to replication stress and DNA damage. In order to better understand the function of this complex, we have studied its interaction with DNA. We find that both ATR and ATRIP associate with chromatin in vivo, and they exist as a large molecular weight complex that can bind single-stranded (ss)DNA cellulose in vitro. Although replication protein A (RPA) is sufficient for the recruitment of ATRIP to ssDNA, we show that a distinct ATR-ATRIP complex is able to bind to DNA with lower affinity in the absence of RPA. In this latter complex, we show that neither ATR nor ATRIP are able to bind DNA individually, nor do they bind DNA in a cooperative manner. However, the addition of HeLa nuclear extract is able to reconstitute the DNA binding of both ATR and ATRIP, suggesting the requirement for an additional protein activity. We also show that ATR is necessary for ATRIP to bind DNA in this low affinity mode and to form a large DNA binding complex. These observations suggest that there are at least two in vitro ATR-ATRIP DNA binding complexes, one which binds DNA with high affinity in an RPA-dependent manner and a second, which binds DNA with lower affinity in an RPA-independent manner but which requires an as of yet unidentified protein.
Bomgarden
Ryan D
RD
Department of Molecular Pharmacology, Stanford University, Stanford, California 94305-5441, USA.
Yean
Dawn
D
Yee
Muh-Ching
MC
Cimprich
Karlene A
KA
D048868:Adaptor Proteins, Signal Transducing; D002460:Cell Line; D002482:Cellulose / Q000235:genetics; D002843:Chromatin / Q000378:metabolism; D004249:DNA Damage / Q000502:physiology; D004260:DNA Repair / Q000502:physiology; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007668:Kidney / Q000166:cytology; D008970:Molecular Weight; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*
C521194:ATRIP protein, human; D048868:Adaptor Proteins, Signal Transducing; D002843:Chromatin; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D010750:Phosphoproteins; D002482:Cellulose; D005090:Exodeoxyribonucleases; C429526:three prime repair exonuclease 1