Determination of deoxyribonucleoside triphosphates using DNA polymerase: a critical evaluation

1981 ◽  
Vol 59 (9) ◽  
pp. 723-727 ◽  
Author(s):  
Darel Hunting ◽  
J. Frank Henderson
Keyword(s):  
Dna Polymerase ◽  
Specific Activity ◽  
Critical Evaluation ◽  
Chinese Hamster ◽  
Dna Polymerase I ◽  
Cell Extracts ◽  
Sensitivity Range ◽  
Deoxyribonucleoside Triphosphate ◽  
Polymerase I

There are more than twenty appreciably different deoxyribonucleoside triphosphate assays using DNA polymerase in the literature. Therefore, each aspect of this method has been critically evaluated, including the purity of the substrates and of DNA polymerase, the reaction conditions, product isolation, and the effect of cell extracts on the assay. The final method uses a phosphatase-free DNA polymerase preparation, 2′-dAMP to inhibit the 3′ → 5′ exonuclease of DNA polymerase I, and includes a correction of both the background incorporation and the sample incorporation for dilution of the specific activity of the radioactive precursor by the sample. The sensitivity, range, accuracy, and reproducibility are reported as well as values for the deoxyribonucleoside triphosphate content of Chinese hamster ovary K-1 cells.

scholarly journals DNA Polymerase I Is Essential for Growth ofMethylobacterium dichloromethanicum DM4 with Dichloromethane

2000 ◽  
Vol 182 (19) ◽  
pp. 5433-5439 ◽  
Author(s):  
Martin F. Kayser ◽  
Michael T. Stumpp ◽  
Stéphane Vuilleumier
Keyword(s):  
Dna Repair ◽  
Dna Polymerase ◽  
Uv Light ◽  
Polymerase Activity ◽  
Dna Polymerase I ◽  
Gene Encoding ◽  
Cell Extracts ◽  
Dichloromethane Dehalogenase ◽  
Polymerase I ◽  
Methylobacterium Dichloromethanicum

ABSTRACT Methylobacterium dichloromethanicum DM4 grows with dichloromethane as the unique carbon and energy source by virtue of a single enzyme, dichloromethane dehalogenase–glutathioneS-transferase. A mutant of the dichloromethane-degrading strain M. dichloromethanicum DM4, strain DM4-1445, was obtained by mini-Tn5 transposon mutagenesis that was no longer able to grow with dichloromethane. Dichloromethane dehalogenase activity in this mutant was comparable to that of the wild-type strain. The site of mini-Tn5 insertion in this mutant was located in the polA gene encoding DNA polymerase I, an enzyme with a well-known role in DNA repair. DNA polymerase activity was not detected in cell extracts of the polA mutant. Conjugation of a plasmid containing the intact DNA polymerase I gene into thepolA mutant restored growth with dichloromethane, indicating that the polA gene defect was responsible for the observed lack of growth of this mutant with dichloromethane. Viability of the DM4-1445 mutant was strongly reduced upon exposure to both UV light and dichloromethane. The polA′-lacZtranscriptional fusion resulting from mini-Tn5 insertion was constitutively expressed at high levels and induced about twofold after addition of 10 mM dichloromethane. Taken together, these data indicate that DNA polymerase I is essential for growth of M. dichloromethanicum DM4 with dichloromethane and further suggest an important role of the DNA repair machinery in the degradation of halogenated, DNA-alkylating compounds by bacteria.

scholarly journals Characteristics of DNA polymerase I from an extreme thermophile, Thermus scotoductus strain K1

2021 ◽  
Vol 10 (1) ◽  
Author(s):  
Ani Saghatelyan ◽  
Hovik Panosyan ◽  
Armen Trchounian ◽  
Nils‐Kåre Birkeland
Keyword(s):  
Dna Polymerase ◽  
Extreme Thermophile ◽  
Dna Polymerase I ◽  
Polymerase I

Site-specific modification of E. coli DNA polymerase I large fragment with pyridoxal 5'-phosphate

Biochemistry ◽  
1984 ◽  
Vol 23 (9) ◽  
pp. 2073-2078 ◽  
Author(s):  
Anup K. Hazra ◽  
Sevilla Detera-Wadleigh ◽  
Samuel H. Wilson
Keyword(s):  
Dna Polymerase ◽  
Large Fragment ◽  
Dna Polymerase I ◽  
Site Specific ◽  
E Coli ◽  
Specific Modification ◽  
Polymerase I
Sign in / Sign up

Export Citation Format

Share Document