scholarly journals Deoxyribonucleic acid polymerases of BHK-21/C13 cells. Partial purification and characterization of the enzymes

1975 ◽  
Vol 145 (2) ◽  
pp. 215-224 ◽  
Author(s):  
R K Craig ◽  
H M Keir
Keyword(s):  
Dna Polymerase ◽  
Extraction Procedure ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Partial Purification ◽  
Dna Polymerase I ◽  
Ph Optimum ◽  
Simple Extraction ◽  
Polymerase I ◽  
Dna Polymerase Ii

DNA polymerase from BHK-21/C13 cells were separated into two species, DNA polymerase I corresponding to the heterogeneous enzyme with sedimentation coefficient of 6-8S, and DNA polymerase II, corresponding to the enzyme with sedimentation coefficient of 3.3S. DNA polymerase I was purified 114-fold and DNA polymerase II 154-fold by a simple extraction procedure followed by column chromatography on phosphocellulose and gel filtration through Sephadex G-100. The purified enzymes differed markedly in respect of pH optimum, stimulation and inhibition by K+, Km for the deoxyribonucleoside 5′-triphosphates, stability to heating at 45 degrees C, and inhibition by N-ethylmaleimide. The preferred primer-template for both enzymes was “activated” DNA (DNA submitted to limited degradation by pancreatic deoxyribonuclease); native or thermally denatured DNA templates were relatively very poorly copied. When certain synthetic templates were tested, substantial differences were revealed between the two enzymes. Poly[d(A-T)] was poorly used by polymerase I but was superior to “activated” DNA for polymerase II. Poly[d(A)]-oligo[d(pT)10] was used efficiently by polymerase I but not by polymerase II. Poly(A)-oligo[d(pT)10] was not an effective primer-template although polymerase I could use it to a limited extent when Mn2+ replaced Mg2+ in the polymerase reaction and when the temperature of incubation was lowered from 37 degrees to 30 degrees C. When only one or two or three triphosphates were supplied in the reaction mixture, the activity of polymerase I was more severly diminished than that of polymerase II.

scholarly journals Deoxyribonucleic acid poymerase of BHK-21/C13 cells. Heterogeneity, molecular asymmetry and subcellular distribution of the enzymes

1975 ◽  
Vol 145 (2) ◽  
pp. 225-232 ◽  
Author(s):  
R K Craig ◽  
H M Keir
Keyword(s):  
Dna Polymerase ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Dna Polymerase I ◽  
Sucrose Density Gradient Centrifugation ◽  
Molecular Weights ◽  
Molecular Asymmetry ◽  
Polymerase I ◽  
Dna Polymerase Ii

Nuclear and cytoplasmic fractions were prepared from exponentially-growing BHK-21/C13 cells; DNA polymerase was extracted from them and analysed by gel filtration and sucrose-density-gradient centrifugation. DNA polymerase I is heterogeneous comprising species covering a considerable range of molecular weights. These have been tentatively identified as four subspecies of apparent molecular weights 900000-1000000 (IA), 460000-560000 (IB), 270000-320000 (IC) and 140000-200000 (ID), as assessed by gel filtration through Sepharose 6B. DNA polymerase II has a mol.wt. of 46000 +/- 4000 as assessed by gel filtration on Sepharose 6B, and 48000 +/- 2000 as assessed by gel filtration on Sephadex G-100. Sedimentation analyses on sucrose density gradients showed that the DNA polymerase I species had sedimentation coefficients predominantly in the range 6-8 S. DNA polymerase II had predominantly a sedimentation coefficient of 3.2 S although a component with lower sedimentation coefficient was found. The lack of correlation between the molecular weights derived from gel filtration and the sedimentation coefficients is attributed to molecular asymmetry. DNA polymerase I was found to be associated predominantly with the cytoplasm although certain types of nuclear preparation contained large amounts of it. DNA polymerase II was found to be mostly if not exclusively in nuclear preparations.

scholarly journals DNA polymerase I is required for premeiotic DNA replication and sporulation but not for X-ray repair in Saccharomyces cerevisiae

1989 ◽  
Vol 9 (2) ◽  
pp. 365-376
Author(s):  
M E Budd ◽  
K D Wittrup ◽  
J E Bailey ◽  
J L Campbell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Temperature Sensitive ◽  
Dna Polymerase I ◽  
X Ray ◽  
Temperature Sensitive Mutants ◽  
Polymerase I ◽  
Dna Polymerase Ii

We have used a set of seven temperature-sensitive mutants in the DNA polymerase I gene of Saccharomyces cerevisiae to investigate the role of DNA polymerase I in various aspects of DNA synthesis in vivo. Previously, we showed that DNA polymerase I is required for mitotic DNA replication. Here we extend our studies to several stages of meiosis and repair of X-ray-induced damage. We find that sporulation is blocked in all of the DNA polymerase temperature-sensitive mutants and that premeiotic DNA replication does not occur. Commitment to meiotic recombination is only 2% of wild-type levels. Thus, DNA polymerase I is essential for these steps. However, repair of X-ray-induced single-strand breaks is not defective in the DNA polymerase temperature-sensitive mutants, and DNA polymerase I is therefore not essential for repair of such lesions. These results suggest that DNA polymerase II or III or both, the two other nuclear yeast DNA polymerases for which roles have not yet been established, carry out repair in the absence of DNA polymerase I, but that DNA polymerase II and III cannot compensate for loss of DNA polymerase I in meiotic replication and recombination. These results do not, however, rule out essential roles for DNA polymerase II or III or both in addition to that for DNA polymerase I.

scholarly journals DNA polymerase I is required for premeiotic DNA replication and sporulation but not for X-ray repair in Saccharomyces cerevisiae.

1989 ◽  
Vol 9 (2) ◽  
pp. 365-376 ◽  
Author(s):  
M E Budd ◽  
K D Wittrup ◽  
J E Bailey ◽  
J L Campbell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Temperature Sensitive ◽  
Dna Polymerase I ◽  
X Ray ◽  
Temperature Sensitive Mutants ◽  
Polymerase I ◽  
Dna Polymerase Ii

We have used a set of seven temperature-sensitive mutants in the DNA polymerase I gene of Saccharomyces cerevisiae to investigate the role of DNA polymerase I in various aspects of DNA synthesis in vivo. Previously, we showed that DNA polymerase I is required for mitotic DNA replication. Here we extend our studies to several stages of meiosis and repair of X-ray-induced damage. We find that sporulation is blocked in all of the DNA polymerase temperature-sensitive mutants and that premeiotic DNA replication does not occur. Commitment to meiotic recombination is only 2% of wild-type levels. Thus, DNA polymerase I is essential for these steps. However, repair of X-ray-induced single-strand breaks is not defective in the DNA polymerase temperature-sensitive mutants, and DNA polymerase I is therefore not essential for repair of such lesions. These results suggest that DNA polymerase II or III or both, the two other nuclear yeast DNA polymerases for which roles have not yet been established, carry out repair in the absence of DNA polymerase I, but that DNA polymerase II and III cannot compensate for loss of DNA polymerase I in meiotic replication and recombination. These results do not, however, rule out essential roles for DNA polymerase II or III or both in addition to that for DNA polymerase I.

scholarly journals Dexyribonucleic acid polymerases of BHK-21/C13cells. Relationship to the physiological state of the cells, and to synchronous indution of synthesis of deoxyribonuleic acid

1975 ◽  
Vol 145 (2) ◽  
pp. 233-240 ◽  
Author(s):  
R K Craig ◽  
P A Costello ◽  
H M Keir
Keyword(s):  
Molecular Weight ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Physiological State ◽  
Polymerase Activity ◽  
Quiescent State ◽  
Dna Polymerase I ◽  
Quiescent Cells ◽  
Polymerase I ◽  
Dna Polymerase Ii

BHK-21/C13 cells were grown in culture under conditions that provided exponentially growing cells and quiescent cells, by modifying the concentration of serum in the growth medium. The high-molecular-weight DNA polymerase (DNA polymerase I) from exponentially growing cells accounted for 90% of the total polymerase activity; the low-molecular-weight DNA polymerase (DNA polymerase II) accounted for the remaining 10%. In quiescent cells, DNA polymerase I contributed only 39% of the total polymerase activity and DNA polymerase II 61%. The total amount of DNA polymerase I in exponentially growing cells was 11.3-fold greater than that in quiescent cells, whereas the amount of DNA polymerase II appeared to be relatively independent of the physiological state of the cells. In an extension of these experiments, cells in a quiescent state (Go cells) were stimulated by the ‘serum-step-up’ method of Burk (1970) to grow and to enter a synchronous wave of DNA synthesis (S-phase cells), 87% of the cells synthesizing DNA at 20 h after the ‘serum-step-up’. During the synchrony experiment, the total cytoplasmic and total nuclear DNA polymerase activities each increased about 4-fold in parallel with the increase in the rate of DNA synthesis. Cytoplasmic polymerase activity was always greater than nuclear polymerase activity. The increases observed were maximal at 20 h after ‘serum step-up’. By 26 h, there was a decrease in enzyme activity (8% for cytoplasmic polymerase and 16% for nuclear polymerase, both relative to the maximum at 20 h), but the rate of DNA synthesis had declined by 37% relative to the maximum at 20 h. In Go cells, DNA polymerase II (mol.wt. 46000 +/- 4000) was the predominant species, there being twice as much of it as of the total DNA polymerase I. In these cells there was little DNA polymerase IC and ID; the amounts of IA (mol.wt. 900 times 10(3)-1100 times 10(3)) and IB (mol.wt. 460 times 10(3)-560 times 10(3)) were about equal but small.

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
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