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

1997 ◽  
Vol 325 (3) ◽  
pp. 815-815
Author(s):  
V. S. KRAYNOV ◽  
B. G. WERNEBURG ◽  
X. ZHONG ◽  
H. LEE ◽  
J. AHN ◽  
...  
Keyword(s):  
Steady State ◽  
Dna Replication ◽  
Substrate Specificity ◽  
Dna Polymerase ◽  
Dna Polymerase Β ◽  
Steady State Kinetics

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

1997 ◽  
Vol 323 (1) ◽  
pp. 103-111 ◽  
Author(s):  
Vadim S. KRAYNOV ◽  
Brian G. WERNEBURG ◽  
Xuejun ZHONG ◽  
Hui LEE ◽  
Jinwoo AHN ◽  
...  
Keyword(s):  
Steady State ◽  
Substrate Specificity ◽  
Dna Polymerase ◽  
Excision Repair ◽  
Catalytic Efficiency ◽  
Main Function ◽  
Wild Type ◽  
Active Site Residues ◽  
Dna Polymerase Β ◽  
Steady State Kinetics

DNA polymerase β (pol β) 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 β, 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 β. (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 β. Upon mutation to give N279A and N279Q, the Kd(app) for the correct dNTP increased by a factor of 15-25. As a consequence, the Kd(app) values for the correct and incorrect nucleotides were similar for N279A and N279Q, suggesting that the main function of the side chain of Asn-279 is in discrimination between the binding of correct and incorrect dNTPs. (iv) In the case of the Y271A mutant, the fidelity and the catalytic efficiency kpol/Kd(app) were little perturbed relative to the wild type. However, both the kpol and Kd(app) values for dNTP were 4-8 times lower in the case of the Y271A mutant than the corresponding values for wild-type pol β. Since the chemical step may not be rate-limiting for wild-type pol β, the effect on kpol could be quite significant if it is caused by a perturbation in the chemical step. (v) Pol β displayed the greatest specificity towards the G:C base pair, which is incorporated during base excision repair of G:U and G:T mispairs. This specificity was slightly enhanced for the Y271F mutant.

DNA Polymerase β:  Pre-Steady-State Kinetic Analysis and Roles of Arginine-283 in Catalysis and Fidelity†

Biochemistry ◽  
1996 ◽  
Vol 35 (22) ◽  
pp. 7041-7050 ◽  
Author(s):  
Brian G. Werneburg ◽  
Jinwoo Ahn ◽  
Xuejun Zhong ◽  
Robert J. Hondal ◽  
Vadim S. Kraynov ◽  
...  
Keyword(s):  
Steady State ◽  
Kinetic Analysis ◽  
Dna Polymerase ◽  
Dna Polymerase Β ◽  
Steady State Kinetic ◽  
State Kinetic

Involvement of DNA polymerase β in DNA replication and mutagenic consequences

2002 ◽  
Vol 315 (5) ◽  
pp. 1039-1047 ◽  
Author(s):  
Laurence Servant ◽  
Anne Bieth ◽  
Hiroshi Hayakawa ◽  
Christophe Cazaux ◽  
Jean-Sébastien Hoffmann
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Dna Polymerase Β

scholarly journals Pre-Steady-State Kinetic Analysis of the Incorporation of Anti-HIV Nucleotide Analogs Catalyzed by Human X- and Y-Family DNA Polymerases

2010 ◽  
Vol 55 (1) ◽  
pp. 276-283 ◽  
Author(s):  
Jessica A. Brown ◽  
Lindsey R. Pack ◽  
Jason D. Fowler ◽  
Zucai Suo
Keyword(s):  
Mitochondrial Dna ◽  
Steady State ◽  
Substrate Specificity ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Steady State Kinetic ◽  
Y Family ◽  
State Kinetic

ABSTRACTNucleoside reverse transcriptase inhibitors (NRTIs) are an important class of antiviral drugs used to manage infections by human immunodeficiency virus, which causes AIDS. Unfortunately, these drugs cause unwanted side effects, and the molecular basis of NRTI toxicity is not fully understood. Putative routes of NRTI toxicity include the inhibition of human nuclear and mitochondrial DNA polymerases. A strong correlation between mitochondrial toxicity and NRTI incorporation catalyzed by human mitochondrial DNA polymerase has been established bothin vitroandin vivo. However, it remains to be determined whether NRTIs are substrates for the recently discovered human X- and Y-family DNA polymerases, which participate in DNA repair and DNA lesion bypassin vivo. Using pre-steady-state kinetic techniques, we measured the substrate specificity constants for human DNA polymerases β, λ, η, ι, κ, and Rev1 incorporating the active, 5′-phosphorylated forms of tenofovir, lamivudine, emtricitabine, and zidovudine. For the six enzymes, all of the drug analogs were incorporated less efficiently (40- to >110,000-fold) than the corresponding natural nucleotides, usually due to a weaker binding affinity and a slower rate of incorporation for the incoming nucleotide analog. In general, the 5′-triphosphate forms of lamivudine and zidovudine were better substrates than emtricitabine and tenofovir for the six human enzymes, although the substrate specificity profile depended on the DNA polymerase. Our kinetic results suggest NRTI insertion catalyzed by human X- and Y-family DNA polymerases is a potential mechanism of NRTI drug toxicity, and we have established a structure-function relationship for designing improved NRTIs.

DNA Polymerase β Fidelity:  Halomethylene-Modified Leaving Groups in Pre-Steady-State Kinetic Analysis Reveal Differences at the Chemical Transition State†

Biochemistry ◽  
2008 ◽  
Vol 47 (3) ◽  
pp. 870-879 ◽  
Author(s):  
Christopher A. Sucato ◽  
Thomas G. Upton ◽  
Boris A. Kashemirov ◽  
Jorge Osuna ◽  
Keriann Oertell ◽  
...  
Keyword(s):  
Steady State ◽  
Transition State ◽  
Kinetic Analysis ◽  
Dna Polymerase ◽  
Dna Polymerase Β ◽  
Steady State Kinetic ◽  
Leaving Groups ◽  
State Kinetic
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