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J. Biol. Chem., Vol. 282, Issue 5, 3357-3366, February 2, 2007

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Interactions of Human O⁶-Alkylguanine-DNA Alkyltransferase (AGT) with Short Single-stranded DNAs^*

Joseph J. Rasimas¹, Sambit R. Kar², Anthony E. Pegg, and Michael G. Fried³

From the Department of Molecular Physiology, Penn State University College of Medicine, Hershey, Pennsylvania 17033 and Department of Molecular and Cellular Biochemistry and Center for Structural Biology, University of Kentucky, Lexington, Kentucky 40536

The O⁶-alkylguanine-DNA alkyltransferase (AGT) repairs O⁶-alkylguanine and O⁴-alkylthymine adducts in single-stranded and duplex DNAs. Here we characterize the binding of AGT to single-stranded DNAs ranging in length from 5 to 78 nucleotides (nt). Binding is moderately cooperative (37.9 ± 3.0 89.8 ± 8.9), resulting in an all-or-nothing association pattern on short templates. This cooperativity contrasts with the isolated binding seen in recent crystal structures of AGT-DNA complexes. The statistical binding site size S (mean = 5.2 ± 0.1) oscillates with increasing template length. The oscillation period (4.10 ± 0.02 nt/protein) is nearly identical to the binding site size obtained at the highest known binding density (S = 4 nt/protein) and is significantly smaller than the contour length (8 bp) occupied in crystalline complexes. A model in which AGT proteins overlap along the DNA contour is proposed to account for these features. Oscillations in intrinsic binding constant K_i and cooperativity factor have the same frequency but are of opposite phase to S with the result that the most stable protein-protein and protein-DNA interactions occur at the highest packing densities. We hypothesize that modest binding cooperativity and high binding densities are adaptations that allow AGT to efficiently search for lesions in the context of chromatin remodeling and DNA replication.

Received for publication, September 15, 2006 , and in revised form, November 3, 2006.

^* This work was supported by National Institutes of Health Grants GM-070662 (to M. G. F.) and CA-97209 (to A. E. P.) and Medical Scientist Training Program Grant 5 T32 GM-08601-05 (to J. J. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Dept. of Psychiatry and Psychology, Mayo Clinic, Rochester, MN 55905.

² Present address: Jules Stein Eye Inst., David Geffen School of Medicine, University of California, Los Angeles, CA 90095.

³ To whom correspondence should be addressed: Dept. of Molecular and Cellular Biochemistry, University of Kentucky, 741 South Limestone, Lexington, KY 40536-0509. Tel.: 859-323-1205; Fax: 859-323-1037; E-mail: michael.fried{at}uky.edu.

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				J. Hu, A. Ma, and A. R. Dinner A two-step nucleotide-flipping mechanism enables kinetic discrimination of DNA lesions by AGT PNAS, March 25, 2008; 105(12): 4615 - 4620. [Abstract] [Full Text] [PDF]