The mutagenic and cytotoxic effects of many alkylating agents are reduced by O6-alkylguanine-DNA alkyltransferase (AGT). In humans, this protein not only protects the integrity of the genome, but also contributes to the resistance of tumors to DNA-alkylating chemotherapeutic agents. Here we describe and test models for cooperative multiprotein complexes of AGT with single-stranded and duplex DNAs that are based on in vitro binding data and the crystal structure of a 1:1 AGT-DNA complex. These models predict that cooperative assemblies contain a three-start helical array of proteins with dominant protein-protein interactions between the amino-terminal face of protein n and the carboxy-terminal face of protein n + 3, and they predict that binding duplex DNA does not require large changes in B-form DNA geometry. Experimental tests using protein cross-linking analyzed by mass spectrometry, electrophoretic and analytical ultracentrifugation binding assays, and topological analyses with closed circular DNA show that the properties of multiprotein AGT-DNA complexes are consistent with these predictions.
|Number of pages||16|
|Journal||Journal of Molecular Biology|
|State||Published - Jun 5 2009|
Bibliographical noteFunding Information:
Mass spectrometric analyses were performed at the University of Kentucky Center for Structural Biology Protein Core Facility. This facility is supported in part by funds from NIH National Center for Research Resources (NCRR) grant P20 RR020171. We gratefully acknowledge the help of Dr. Carol Beach in acquiring these data. Research in this report was supported by NIH grants GM-070662 (to M.G.F.), CA-018137 and CA-097209 (to A.E.P.), NS-38041, DA-02243, and RR-20171 (to D.W.R.), and Medical Scientist Training Program grant 5 T32 GM-08601-05 (to J.J.R.).
- DNA repair
- O-alkylguanine-DNA alkyltransferase
- binding cooperativity
- methylguanine methyltransferase
- protein contacts
ASJC Scopus subject areas
- Structural Biology
- Molecular Biology