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J Biol Chem, Vol. 273, Issue 18, 11121-11126, May 1, 1998

Functional Analysis of the Amino-terminal 8-kDa Domain of DNA Polymerase  as Revealed by Site-directed Mutagenesis
DNA BINDING AND 5'-DEOXYRIBOSE PHOSPHATE LYASE ACTIVITIES

Rajendra Prasad^**, William A. Beard^**, Jan Y. Chyan^**, Mark W. Maciejewski^¶, Gregory P. Mullen^¶, and Samuel H. Wilson^**

From the  Laboratory of Structural Biology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, ^** Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555, and ^¶ Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06032

The amino-terminal 8-kDa domain of DNA polymerase  functions in binding single-stranded DNA (ssDNA), recognition of a 5'-phosphate in gapped DNA structures, and as a 5'-deoxyribose phosphate (dRP) lyase. NMR and x-ray crystal structures of this domain have suggested several residues that may interact with ssDNA or play a role in the dRP lyase reaction. Nine of these residues were altered by site-directed mutagenesis. Each mutant was expressed in Escherichia coli, and the recombinant protein was purified to near homogeneity. CD spectra of these mutant proteins indicated that the alteration did not adversely affect the global protein structure. Single-stranded DNA binding was probed by photochemical cross-linking to oligo(dT)₁₆. Several mutants (F25W, K35A, K60A, and K68A) were impaired in ssDNA binding activity, whereas other mutants (H34G, E71Q, K72A, E75A, and K84A) retained near wild-type binding activity. The 5'-phosphate recognition activity of these mutants was examined by UV cross-linking to a 5-nucleotide gap DNA where the 5' terminus in the gap was either phosphorylated or unphosphorylated. The results indicate that Lys³⁵ is involved in 5'-phosphate recognition of DNA polymerase . Finally, the dRP lyase activity of these mutants was evaluated using a preincised apurinic/apyrimidinic DNA. Alanine mutants of Lys³⁵ and Lys⁶⁰ are significantly reduced in dRP lyase activity, consistent with the lower ssDNA binding activity. More importantly, alanine substitution for Lys⁷² resulted in a greater than 90% loss of dRP lyase activity, without affecting DNA binding. Alanine mutants of Lys⁶⁸ and Lys⁸⁴ had wild-type dRP lyase activity. The triple alanine mutant, K35A/K68A/K72A, was devoid of dRP lyase activity, suggesting that the effects of the alanine substitution at Lys⁷² and Lys³⁵ were additive. The results suggest that Lys⁷² is directly involved in formation of a covalent imino intermediate and are consistent with Lys⁷² as the predominant Schiff base nucleophile in the dRP lyase -elimination catalytic reaction.

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