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 Crystal structures of DNA polymerase I capture novel intermediates in the DNA synthesis pathway

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Nicholas Chim ◽  
Lynnette N Jackson ◽  
Anh M Trinh ◽  
John C Chaput
Keyword(s):  
High Resolution ◽  
Dna Synthesis ◽  
Crystal Structures ◽  
Dna Polymerase ◽  
Active Site ◽  
Dna Polymerase I ◽  
Dynamic Nature ◽  
Enzyme Active Site ◽  
Polymerase I ◽  
In Cells

High resolution crystal structures of DNA polymerase intermediates are needed to study the mechanism of DNA synthesis in cells. Here we report five crystal structures of DNA polymerase I that capture new conformations for the polymerase translocation and nucleotide pre-insertion steps in the DNA synthesis pathway. We suggest that these new structures, along with previously solved structures, highlight the dynamic nature of the finger subdomain in the enzyme active site.

Inhibition by Cyclic Guanosine 3':5'-Monophosphate of the Soluble DNA Polymerase Activity, and of Partially Purified DNA Polymerase A (DNA Polymerase I) from the Yeast Saccharomyces cerevisiae

1981 ◽  
Vol 36 (9-10) ◽  
pp. 813-819 ◽  
Author(s):  
Hans Eckstein
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerases ◽  
Nuclease Activity ◽  
Polymerase Activity ◽  
Yeast Cells ◽  
Primer Binding Site ◽  
Dna Polymerase I ◽  
Yeast Saccharomyces Cerevisiae ◽  
Main Component ◽  
Polymerase I

Abstract Dedicated to Professor Dr. Joachim Kühnau on the Occasion of His 80th Birthday cGMP, DNA Polymerase Activity, DNA Polymerase A, DNA Polymerase I, Baker's Yeast DNA polymerase activity from extracts of growing yeast cells is inhibited by cGMP. Experiments with partially purified yeast DNA polymerases show, that cGMP inhibits DNA polymerase A (DNA polymerase I from Chang), which is the main component of the soluble DNA polymerase activity in yeast extracts, by competing for the enzyme with the primer-template DNA. Since the enzyme is not only inhibited by 3',5'-cGMP, but also by 3',5'-cAMP, the 3': 5'-phosphodiester seems to be crucial for the competition between cGMP and primer. This would be inconsistent with the concept of a 3'-OH primer binding site in the enzyme. The existence of such a site in the yeast DNA polymerase A is indicated from studies with various purine nucleoside monophosphates.When various DNA polymerases are compared, inhibition by cGMP seems to be restricted to those enzymes, which are involved in DNA replication. DNA polymerases with an associated nuclease activity are not inhibited, DNA polymerase B from yeast is even activated by cGMP. Though some relations between the cGMP effect and the presumed function of the enzymes in the living cell are apparent, the biological meaning of the observations in general remains open.

scholarly journals Understanding the Effect of Multiple Domain Deletion in DNA Polymerase I from Geobacillus Sp. Strain SK72

Catalysts ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 936
Author(s):  
Waqiyuddin Hilmi Hadrawi ◽  
Anas Norazman ◽  
Fairolniza Mohd Shariff ◽  
Mohd Shukuri Mohamad Ali ◽  
Raja Noor Zaliha Raja Abd Rahman
Keyword(s):  
Dna Polymerase ◽  
Polymerase Activity ◽  
Functional Domain ◽  
Negative Effects ◽  
Dna Polymerase I ◽  
Ph Profile ◽  
Structural Support ◽  
Exonuclease Domain ◽  
Polymerase I ◽  
Multiple Domain

The molecular structure of DNA polymerase I or family A polymerases is made up of three major domains that consist of a single polymerase domain with two extra exonuclease domains. When the N-terminal was deleted, the enzyme was still able to perform basic polymerase activity with additional traits that used isothermal amplification. However, the 3′-5′ exonuclease domain that carries a proofreading activity was disabled. Yet, the structure remained attached to the 5′-3′ polymerization domain without affecting its ability. The purpose of this non-functional domain still remains scarce. It either gives negative effects or provides structural support to the DNA polymerase. Here, we compared the effect of deleting each domain against the polymerase activity. The recombinant wild type and its variants were successfully purified and characterized. Interestingly, SK72-Exo (a large fragment excluding the 5′-3′ exonuclease domain) exhibited better catalytic activity than the native SK72 (with all three domains) at similar optimum temperature and pH profile, and it showed longer stability at 70 °C. Meanwhile, SK72-Exo2 (polymerization domain without both the 5′-3′ and 3′-5′ exonuclease domain) displayed the lowest activity with an optimum at 40 °C and favored a more neutral environment. It was also the least stable among the variants, with almost no activity at 50 °C for the first 10 min. In conclusion, cutting both exonuclease domains in DNA polymerase I has a detrimental effect on the polymerization activity and structural stability.

Mechanism of initiation of in vitro DNA synthesis by the immunopurified complex between yeast DNA polymerase I and DNA primase

1986 ◽  
Vol 161 (2) ◽  
pp. 435-440 ◽  
Author(s):  
Gianfranco BADARACCO ◽  
Paola VALSASNINI ◽  
Marco FOIANI ◽  
Roberta BENFANTE ◽  
Giovanna LUCCHINI ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Dna Polymerase I ◽  
Dna Primase ◽  
Polymerase I

scholarly journals DNA polymerases, deoxyribonucleases, and recombination during meiosis in Saccharomyces cerevisiae.

1984 ◽  
Vol 4 (12) ◽  
pp. 2811-2817 ◽  
Author(s):  
M A Resnick ◽  
A Sugino ◽  
J Nitiss ◽  
T Chow
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
High Efficiency ◽  
Intragenic Recombination ◽  
Dna Polymerase I ◽  
Crude Extracts ◽  
Total Dna ◽  
Polymerase I ◽  
Kinetics Of

We utilized strains of Saccharomyces cerevisiae that exhibit high efficiency of synchrony of meiosis to examine several aspects of meiosis including sporulation, recombination, DNA synthesis, DNA polymerase I and II, and Mg2+-dependent alkaline DNases. The kinetics of commitment to intragenic recombination and sporulation are similar. The synthesis of DNA, as measured directly with diphenylamine, appears to precede the commitment to recombination. Both DNA polymerase I and II activities and total DNA-synthesizing activity in crude extracts increase two- to threefold before the beginning of meiotic DNA synthesis. Increases of 10- to 20-fold over mitotic levels are found for Mg2+-dependent alkaline DNase activity in crude extracts before and during the commitment to meiotic intragenic recombination. Of particular interest is the comparable increase in a nuclease under the control of the RAD52 gene; this enzyme has been identified by the use of antibody raised against a similar enzyme from Neurospora crassa. Since the RAD52 gene is essential for meiotic recombination, the nuclease is implicated in the high levels of recombination observed during meiosis. The effects observed in this report are meiosis specific since they are not observed in an alpha alpha strain.

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