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.

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 Polymerization activity of an alpha-like DNA polymerase requires a conserved 3'-5' exonuclease active site

1991 ◽  
Vol 11 (9) ◽  
pp. 4786-4795
Author(s):  
J S Gibbs ◽  
K Weisshart ◽  
P Digard ◽  
A deBruynKops ◽  
D M Knipe ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Active Site ◽  
Dna Polymerases ◽  
Polymerase Activity ◽  
Cell Nuclei ◽  
Dna Polymerase I ◽  
Viral Dna ◽  
Gene Encoding ◽  
Simplex Virus ◽  
Polymerase I

Most DNA polymerases are multifunctional proteins that possess both polymerizing and exonucleolytic activities. For Escherichia coli DNA polymerase I and its relatives, polymerase and exonuclease activities reside on distinct, separable domains of the same polypeptide. The catalytic subunits of the alpha-like DNA polymerase family share regions of sequence homology with the 3'-5' exonuclease active site of DNA polymerase I; in certain alpha-like DNA polymerases, these regions of homology have been shown to be important for exonuclease activity. This finding has led to the hypothesis that alpha-like DNA polymerases also contain a distinct 3'-5' exonuclease domain. We have introduced conservative substitutions into a 3'-5' exonuclease active site homology in the gene encoding herpes simplex virus DNA polymerase, an alpha-like polymerase. Two mutants were severely impaired for viral DNA replication and polymerase activity. The mutants were not detectably affected in the ability of the polymerase to interact with its accessory protein, UL42, or to colocalize in infected cell nuclei with the major viral DNA-binding protein, ICP8, suggesting that the mutation did not exert global effects on protein folding. The results raise the possibility that there is a fundamental difference between alpha-like DNA polymerases and E. coli DNA polymerase I, with less distinction between 3'-5' exonuclease and polymerase functions in alpha-like DNA polymerases.

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 Catalytically inactive T7 DNA polymerase imposes a lethal replication roadblock

2020 ◽  
Vol 295 (28) ◽  
pp. 9542-9550
Author(s):  
Alfredo J. Hernandez ◽  
Seung-Joo Lee ◽  
Seungwoo Chang ◽  
Jaehun A. Lee ◽  
Joseph J. Loparo ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Metal Binding ◽  
Polymerase Activity ◽  
Stable Complex ◽  
Klenow Fragment ◽  
Dna Polymerase I ◽  
E Coli ◽  
Growth System ◽  
Polymerase I

Bacteriophage T7 encodes its own DNA polymerase, the product of gene 5 (gp5). In isolation, gp5 is a DNA polymerase of low processivity. However, gp5 becomes highly processive upon formation of a complex with Escherichia coli thioredoxin, the product of the trxA gene. Expression of a gp5 variant in which aspartate residues in the metal-binding site of the polymerase domain were replaced by alanine is highly toxic to E. coli cells. This toxicity depends on the presence of a functional E. coli trxA allele and T7 RNA polymerase-driven expression but is independent of the exonuclease activity of gp5. In vitro, the purified gp5 variant is devoid of any detectable polymerase activity and inhibited DNA synthesis by the replisomes of E. coli and T7 in the presence of thioredoxin by forming a stable complex with DNA that prevents replication. On the other hand, the highly homologous Klenow fragment of DNA polymerase I containing an engineered gp5 thioredoxin-binding domain did not exhibit toxicity. We conclude that gp5 alleles encoding inactive polymerases, in combination with thioredoxin, could be useful as a shutoff mechanism in the design of a bacterial cell-growth system.

scholarly journals Enhancement of Polymerase Activity of the Large Fragment in DNA Polymerase I from Geobacillus stearothermophilus by Site-Directed Mutagenesis at the Active Site

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Yi Ma ◽  
Beilei Zhang ◽  
Meng Wang ◽  
Yanghui Ou ◽  
Jufang Wang ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Rapid Detection ◽  
Polymerase Activity ◽  
Site Directed Mutagenesis ◽  
High Price ◽  
Geobacillus Stearothermophilus ◽  
Large Fragment ◽  
Dna Polymerase I ◽  
Primary Diagnostics ◽  
Polymerase I

The large fragment of DNA polymerase I from Geobacillus stearothermophilus GIM1.543 (Bst DNA polymerase) with 5′-3′ DNA polymerase activity while in absence of 5′-3′ exonuclease activity possesses high thermal stability and polymerase activity. Bst DNA polymerase was employed in isothermal multiple self-matching initiated amplification (IMSA) which amplified the interest sequence with high selectivity and was widely applied in the rapid detection of human epidemic diseases. However, the detailed information of commercial Bst DNA polymerase is unpublished and well protected by patents, which makes the high price of commercial kits. In this study, wild-type Bst DNA polymerase (WT) and substitution mutations for improving the efficiency of DNA polymerization were expressed and purified in E. coli. Site-directed substitutions of four conserved residues (Gly310, Arg412, Lys416, and Asp540) in the activity site of Bst DNA polymerase influenced efficiency of polymerizing dNTPs. The substitution of residue Gly310 by alanine or leucine and residue Asp540 by glutamic acid increased the efficiency of polymerase activity. All mutants with higher polymerizing efficiency were employed to complete the rapid detection of EV71-associated hand, foot, and mouth disease (HFMD) by IMSA approach with relatively shorter period which is suitable for the primary diagnostics setting in rural and underdeveloped areas.

scholarly journals ATP-Stimulated Polymerase Activity Involving DNA Polymerase I and a recB-Dependent Factor in Extracts of Escherichia coli Cells.

1987 ◽  
Vol 41b ◽  
pp. 332-335 ◽  
Author(s):  
Juhani Eerik Syväoja ◽  
Märtha Larsson-Raźnikiewicz ◽  
Benito Rodriguez ◽  
Kwai Ming Cheung ◽  
James H. P. Utley
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Polymerase Activity ◽  
Dna Polymerase I ◽  
Escherichia Coli Cells ◽  
Polymerase I ◽  
Dependent Factor
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