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Molecular and Cellular Biology, November 2003, p. 8316-8322, Vol. 23, No. 22
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.22.8316-8322.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

The Mechanism of Nucleotide Incorporation by Human DNA Polymerase Differs from That of the Yeast Enzyme

M. Todd Washington, Robert E. Johnson, Louise Prakash, and Satya Prakash^*

Sealy Center for Molecular Science, University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1061

Received 3 July 2003/ Accepted 11 August 2003

DNA polymerase (Pol) catalyzes the efficient and accurate synthesis of DNA opposite cyclobutane pyrimidine dimers, and inactivation of Pol in humans causes the cancer-prone syndrome, the variant form of xeroderma pigmentosum. Pre-steady-state kinetic studies of yeast Pol have indicated that the low level of fidelity of this enzyme results from a poorly discriminating induced-fit mechanism. Here we examine the mechanistic basis of the low level of fidelity of human Pol. Because the human and yeast enzymes behave similarly under steady-state conditions, we expected these enzymes to utilize similar mechanisms of nucleotide incorporation. Surprisingly, however, we find that human Pol differs from the yeast enzyme in several important respects. The human enzyme has a 50-fold-faster rate of nucleotide incorporation than the yeast enzyme but binds the nucleotide with an approximately 50-fold-lower level of affinity. This lower level of binding affinity might provide a means of regulation whereby the human enzyme remains relatively inactive except when the cellular deoxynucleoside triphosphate concentrations are high, as may occur during DNA damage, thereby avoiding the mutagenic consequences arising from the inadvertent action of this enzyme during normal DNA replication.

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* Corresponding author. Mailing address: Sealy Center for Molecular Science, University of Texas Medical Branch at Galveston, 6.104 Blocker Medical Research Building, 11th and Mechanic Streets, Galveston, TX 77555-1061. Phone: (409) 747-8602. Fax: (409) 747-8608. E-mail: s.prakash{at}utmb.edu.

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Molecular and Cellular Biology, November 2003, p. 8316-8322, Vol. 23, No. 22
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.22.8316-8322.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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