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Evidence for a Watson-Crick Hydrogen Bonding Requirement in DNA Synthesis by Human DNA Polymerase

William T. Wolfle,¹ M. Todd Washington,^1, Eric T. Kool,² Thomas E. Spratt,³ Sandra A. Helquist,² Louise Prakash,¹ and Satya Prakash^1*

Sealy Center for Molecular Science, University of Texas Medical Branch at Galveston, 6.104 Blocker Medical Research Building, 11th and Mechanic Streets, Galveston, Texas 77555-1061,¹ Department of Chemistry, Stanford University, Stanford, California 94305-5080,² American Health Foundation, 1 Dana Road, Valhalla, New York 10595³

Received 6 May 2005/ Returned for modification 24 May 2005/ Accepted 27 May 2005

The efficiency and fidelity of nucleotide incorporation by high-fidelity replicative DNA polymerases (Pols) are governed by the geometric constraints imposed upon the nascent base pair by the active site. Consequently, these polymerases can efficiently and accurately replicate through the template bases which are isosteric to natural DNA bases but which lack the ability to engage in Watson-Crick (W-C) hydrogen bonding. DNA synthesis by Pol, a low-fidelity polymerase able to replicate through DNA lesions, however, is inhibited in the presence of such an analog, suggesting a dependence of this polymerase upon W-C hydrogen bonding. Here we examine whether human Pol, which differs from Pol in having a higher fidelity and which, unlike Pol, is inhibited at inserting nucleotides opposite DNA lesions, shows less of a dependence upon W-C hydrogen bonding than does Pol. We find that an isosteric thymidine analog is replicated with low efficiency by Pol, whereas a nucleobase analog lacking minor-groove H bonding potential is replicated with high efficiency. These observations suggest that both Pol and Pol rely on W-C hydrogen bonding for localizing the nascent base pair in the active site for the polymerization reaction to occur, thus overcoming these enzymes' low geometric selectivity.

Present address: Department of Biochemistry, University of Iowa, 51 Newton Rd., Iowa City, IA 552242-1109.

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