PMID: 9173867Apr 1, 1997Paper

DNA polymerase beta: analysis of the contributions of tyrosine-271 and asparagine-279 to substrate specificity and fidelity of DNA replication by pre-steady-state kinetics

The Biochemical Journal
V S KraynovM D Tsai

Abstract

DNA polymerase beta (pol beta) from rat brain, overexpressed in Escherichia coli, was used as a model to study the factors responsible for substrate specificity [kpol, Kd(app) and kpol/Kd(app)] and fidelity during DNA synthesis. The roles of two active-site residues, Asn-279 and Tyr-271, were examined by construction of N279A, N279Q, Y271A, Y271F and Y271S mutants followed by structural analyses by NMR and CD and functional analyses by pre-steady-state kinetics. The results are summarized as follows. (i) None of the two-dimensional NMR spectra of the mutants was significantly perturbed relative to that for wild-type pol beta, suggesting that Tyr-271 and Asn-279 are not important for the global structure of the protein. (ii) CD analyses of guanidinium hydrochloride-induced denaturation showed that all mutants behaved similarly to the wild type in the free energy of denaturation, suggesting that Tyr-271 and Asn-279 are not critical for the conformational stability of pol beta. (iii) The Kd(app) for the correct dNTP was lower than that for the incorrect dNTP by a factor of 10-30 in the case of wild-type pol beta. Upon mutation to give N279A and N279Q, the Kd(app) for the correct dNTP increased by a factor of 15-25. As a consequenc...Continue Reading

Citations

May 18, 2004·The Journal of Biological Chemistry·William A BeardSamuel H Wilson
Apr 8, 2004·Proceedings of the National Academy of Sciences of the United States of America·Ravi Radhakrishnan, Tamar Schlick
Apr 9, 2008·Proceedings of the National Academy of Sciences of the United States of America·Ping LinLee G Pedersen
Oct 18, 2013·The Journal of Biological Chemistry·Brian E EckenrothSylvie Doublié
Feb 9, 2007·FEBS Letters·Václav MartínekJan Florián
Aug 30, 2006·Proceedings of the National Academy of Sciences of the United States of America·Ping LinLee G Pedersen
May 20, 2005·The Journal of Biological Chemistry·Daniela StarcevicJoann B Sweasy
Oct 3, 2013·Nucleic Acids Research·Patricia GarridoAngel J Picher
Apr 11, 2007·Biochemistry·Marina BakhtinaMing-Daw Tsai
Jul 26, 2006·The Journal of Physical Chemistry. B·Karunesh Arora, Tamar Schlick
Feb 9, 2006·Chemical Reviews·William A Beard, Samuel H Wilson
Feb 3, 2016·Journal of the American Chemical Society·Mu-Sen LiuMing-Daw Tsai
Mar 13, 2014·Journal of the American Chemical Society·Wen-Jin WuMing-Daw Tsai
Jul 9, 2004·Journal of the American Chemical Society·Linjing YangTamar Schlick
Oct 27, 2004·Biochemistry·Michelle P RoettgerZucai Suo
Sep 22, 2005·Journal of the American Chemical Society·Ravi Radhakrishnan, Tamar Schlick
Jul 2, 2009·Parasitology International·Juan A VenegasAldo Solari
Jul 28, 2009·Biochimica Et Biophysica Acta·Jennifer Yamtich, Joann B Sweasy

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