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 Previous Article

Molecular and Cellular Biology, January 2004, p. 936-943, Vol. 24, No. 2
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.2.936-943.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Human DNA Polymerase {iota} Utilizes Different Nucleotide Incorporation Mechanisms Dependent upon the Template Base

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 12 September 2003/ Returned for modification 17 October 2003/ Accepted 22 October 2003

Human DNA polymerase {iota} (Pol{iota}) is a member of the Y family of DNA polymerases involved in translesion DNA synthesis. Pol{iota} is highly unusual in that it possesses a high fidelity on template A, but has an unprecedented low fidelity on template T, preferring to misincorporate a G instead of an A. To understand the mechanisms of nucleotide incorporation opposite different template bases by Pol{iota}, we have carried out pre-steady-state kinetic analyses of nucleotide incorporation opposite templates A and T. These analyses have revealed that opposite template A, the correct nucleotide is preferred because it is bound tighter and is incorporated faster than the incorrect nucleotides. Opposite template T, however, the correct and incorrect nucleotides are incorporated at very similar rates, and interestingly, the greater efficiency of G misincorporation relative to A incorporation opposite T arises predominantly from the tighter binding of G. Based on these results, we propose that the incipient base pair is accommodated differently in the active site of Pol{iota} dependent upon the template base and that when T is the templating base, Pol{iota} accommodates the wobble base pair better than the Watson-Crick base pair.

* 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.

Molecular and Cellular Biology, January 2004, p. 936-943, Vol. 24, No. 2
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.2.936-943.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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