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J. Biol. Chem., Vol. 283, Issue 10, 6154-6161, March 7, 2008

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The Positively Charged Surface of Herpes Simplex Virus UL42 Mediates DNA Binding^*

Gloria Komazin-Meredith, Webster L. Santos¹, David J. Filman, James M. Hogle, Gregory L. Verdine, and Donald M. Coen²

From the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115 and Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138

Herpes simplex virus DNA polymerase is a heterodimer composed of UL30, a catalytic subunit, and UL42, a processivity subunit. Mutations that decrease DNA binding by UL42 decrease long chain DNA synthesis by the polymerase. The crystal structure of UL42 bound to the C terminus of UL30 revealed an extensive positively charged surface ("back face"). We tested two hypotheses, 1) the C terminus of UL30 affects DNA binding and 2) the positively charged back face mediates DNA binding. Addressing the first hypothesis, we found that the presence of a peptide corresponding to the UL30 C terminus did not result in altered binding of UL42 to DNA. Addressing the second hypothesis, previous work showed that substitution of four conserved arginine residues on the basic face with alanines resulted in decreased DNA affinity. We tested the affinities for DNA and the stimulation of long chain DNA synthesis of mutants in which the four conserved arginine residues were substituted individually or together with lysines and also a mutant in which a conserved glutamine residue was substituted with an arginine to increase positive charge on the back face. We also engineered cysteines onto this surface to permit disulfide cross-linking studies. Last, we assayed the effects of ionic strength on DNA binding by UL42 to estimate the number of ions released upon binding. Our results taken together strongly suggest that the basic back face of UL42 contacts DNA and that positive charge on this surface is important for this interaction.

Received for publication, October 19, 2007 , and in revised form, January 4, 2008.

^* This work was supported in part by National Institutes of Health Grants RO1 A119838 and AI26077 (to D. M. C.) and GM 044853 (to G. L. V.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1.

¹ Recipient of a Ruth L. Kirchstein National Research Service Award F32GM067380. Present address: Department of Chemistry, Virginia Tech, 107 Davidson Hall, Blacksburg, VA 24061.

² To whom correspondence should be addressed: Dept. of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Ave., Boston, MA. Fax: 617-432-3833; E-mail: Don_Coen{at}hms.harvard.edu.

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