Recognition of template-primer and gapped DNA substrates by the human DNA polymerase β

2001 ◽  
Vol 308 (3) ◽  
pp. 477-500 ◽  
Author(s):  
Surendran Rajendran ◽  
Maria J. Jezewska ◽  
Wlodzimierz Bujalowski
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerase Β ◽  
Human Dna ◽  
Dna Substrates ◽  
Gapped Dna

scholarly journals Energetics and Specificity of Rat DNA Polymerase β Interactions with Template-primer and Gapped DNA Substrates

2001 ◽  
Vol 276 (19) ◽  
pp. 16123-16136 ◽  
Author(s):  
Maria J. Jezewska ◽  
Surendran Rajendran ◽  
Wlodzimierz Bujalowski
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerase Β ◽  
Dna Substrates ◽  
Gapped Dna

scholarly journals DNA polymerase β: effects of gapped DNA substrates on dNTP specificity, fidelity, processivity and conformational changes1

1998 ◽  
Vol 331 (1) ◽  
pp. 79-87 ◽  
Author(s):  
Jinwoo AHN ◽  
Vadim S. KRAYNOV ◽  
Xuejun ZHONG ◽  
Brian G. WERNEBURG ◽  
Ming-Daw TSAI
Keyword(s):  
Conformational Change ◽  
Dna Polymerase ◽  
Catalytic Efficiency ◽  
Duplex Dna ◽  
Gap Filling ◽  
Dna Polymerase Β ◽  
Base Pairs ◽  
Single Nucleotide ◽  
Dna Substrates ◽  
Gapped Dna

Pre-steady-state kinetic analysis was used to compare the catalytic properties of DNA polymerase β (Pol β) for single-base gap-filling and regular duplex DNA synthesis. The rate of polymerization (kpol) and the apparent equilibrium dissociation constant of dNTP (Kd) were determined with single-nucleotide gapped DNA substrates for all four possible correct base pairs and twelve possible incorrect base pairs, and the results were compared with those obtained previously with non-gapped primer/template duplex DNA substrates. For correct dNTP incorporation, the use of single-nucleotide gapped DNA led to significant decreases in the Kd of dNTP. Although kpol was little affected, the catalytic efficiency kpol/Kd increased significantly owing to the decreases in Kd. In contrast, for incorrect dNTP incorporation, the use of single-nucleotide gapped DNA substrates did not affect the Kd of dNTP appreciably but caused the kpol (and thus kpol/Kd) for incorrect dNTP incorporation to increase. As a consequence the fidelity of Pol β was not significantly affected by the use of single-nucleotide gapped DNA substrates. In addition we show that under processive polymerization conditions the processivity of Pol β increases in the gap-filling synthesis owing to a decreased rate of DNA dissociation. Finally, with a single-nucleotide gapped DNA substrate the rate-limiting conformational change step before chemistry was also observed. However, the preceding fast conformational change observed with duplex DNA substrates was not clearly detected. A possible cause is that in the complex with the gapped DNA, the 8 kDa N-terminal domain of Pol β already exists in a closed conformation. This interpretation was supported by tryptic digestion experiments.

Kinetic study of various binding modes between human DNA polymerase β and different DNA substrates by surface-plasmon-resonance biosensor

2002 ◽  
Vol 361 (2) ◽  
pp. 317-325 ◽  
Author(s):  
Pui Yan TSOI ◽  
Mengsu YANG
Keyword(s):  
Surface Plasmon Resonance ◽  
Dna Polymerase ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Single Strand ◽  
The Other ◽  
Dna Polymerase Β ◽  
Human Dna ◽  
Dna Template ◽  
Gapped Dna

The interaction of a series of DNA substrates with human DNA polymerase β has been studied in real time by using a surface-plasmon-resonance (SPR) biosensor technique. We have prepared the sensor surfaces comprising different DNA targets, including single-stranded DNA, blunt-end double-stranded DNA, gapped DNA and DNA template—primer duplexes containing various mismatches at different positions. The binding and dissociation of polymerase β at the DNA-modified surfaces was measured in real time, and the kinetics profiles of polymerase—DNA interaction were analysed using various physical models. The results showed that polymerase β binding to single-stranded DNA (KA = 1.25×108M−1; where KA is the equilibrium affinity constant) was thermodynamically more favourable than to blunt-end DNA duplex (KA = 7.56×107M−1) or gapped DNA (KA = 8.53×107M−1), with a single binding mode on each DNA substrate. However, polymerase β bound to DNA template—primer duplexes (15bp with a 35nt overhang) at two sites, presumably one at the single-strand overhang and the other at the 3′-end of the primer. When the DNA duplex was fully matched, most of the polymerase β (83%) bound to the template—primer duplex region. The introduction of different numbers of mismatches near the 3′-end of the primer caused the binding affinity and the fraction of polymerase β bound at the duplex region to decrease 8–58-fold and 15–40%, respectively. On the other hand, the affinity of polymerase β for the single-strand overhang remained unchanged while the fraction bound to the single-strand region increased by 15–40%. The destabilizing effect of the mismatches was due to both a decrease in the rate of binding and an increase in the rate of dissociation for polymerase β.

scholarly journals Base excision repair and design of small molecule inhibitors of human DNA polymerase β

2010 ◽  
Vol 67 (21) ◽  
pp. 3633-3647 ◽  
Author(s):  
Samuel H. Wilson ◽  
William A. Beard ◽  
David D. Shock ◽  
Vinod K. Batra ◽  
Nisha A. Cavanaugh ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Small Molecule ◽  
Base Excision Repair ◽  
Small Molecule Inhibitors ◽  
Excision Repair ◽  
Dna Polymerase Β ◽  
Human Dna ◽  
Base Excision

Detection and characterization of DNA polymerase activity in Toxoplasma gondii

Parasitology ◽  
1993 ◽  
Vol 107 (2) ◽  
pp. 135-139 ◽  
Author(s):  
A. Makioka ◽  
B. Stavros ◽  
J. T. Ellis ◽  
A. M. Johnson
Keyword(s):  
Toxoplasma Gondii ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Sedimentation Coefficient ◽  
Polymerase Activity ◽  
Magnesium Ions ◽  
Dna Polymerase Β ◽  
Human Dna ◽  
Approximate Molecular Weight

SUMMARYA DNA polymerase activity has been detected and characterized in crude extracts from tachzoites of Toxoplasma gondii. The enzyme has a sedimentation coefficient of 6·4 S, corresponding to an approximate molecular weight of 150000 assuming a globular shape. Like mammalian DNA polymerase α, the DNA polymerase of T. gondii was sensitive to N-ethylmaleimide and inhibited by high ionic strength. However, the enzyme activity was not inhibited by aphidicolin which is an inhibitor of mammalian DNA polymerases α, δ and ε and also cytosine-β-D-arabinofuranoside-5′-triphosphate which is an inhibitor of α polymerase. The activity was inhibited by 2′,3′-dideoxythymidine-5′-triphosphate which is an inhibitor of mammalian DNA polymerase β and γ. Magnesium ions (Mg2+) were absolutely required for activity and its optimal concentration was 6 mM. The optimum potassium (K+) concentration was 50 mM and a higher concentration of K+ markedly inhibited the activity. Activity was optimal at pH 8. Monoclonal antibodies against human DNA polymerase did not bind to DNA polymerase of T. gondii. Thus the T. gondii enzyme differs from the human enzymes and may be a useful target for the design of toxoplasmacidal drugs.

scholarly journals Thermodynamics of Human DNA Ligase I Trimerization and Association with DNA Polymerase β

1998 ◽  
Vol 273 (32) ◽  
pp. 20540-20550 ◽  
Author(s):  
Emilios K. Dimitriadis ◽  
Rajendra Prasad ◽  
Mary K. Vaske ◽  
Ling Chen ◽  
Alan E. Tomkinson ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Dna Ligase ◽  
Dna Polymerase Β ◽  
Human Dna ◽  
Dna Ligase I
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