Mutability of DNA polymerase I: Implications for the creation of mutant DNA polymerases

DNA Repair ◽  
2005 ◽  
Vol 4 (12) ◽  
pp. 1390-1398 ◽  
Author(s):  
Ern Loh ◽  
Lawrence A. Loeb
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerases ◽  
Dna Polymerase I ◽  
The Creation ◽  
Polymerase I

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 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 Rapid Identification of Genes Encoding DNA Polymerases by Function-Based Screening of Metagenomic Libraries Derived from Glacial Ice

2009 ◽  
Vol 75 (9) ◽  
pp. 2964-2968 ◽  
Author(s):  
Carola Simon ◽  
Judith Herath ◽  
Stephanie Rockstroh ◽  
Rolf Daniel
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Rapid Identification ◽  
Dna Polymerase I ◽  
Glacial Ice ◽  
Metagenomic Libraries ◽  
Genes Encoding ◽  
Polymerase I ◽  
Encoding Genes

ABSTRACT Small-insert and large-insert metagenomic libraries were constructed from glacial ice of the Northern Schneeferner, which is located on the Zugspitzplatt in Germany. Subsequently, these libraries were screened for the presence of DNA polymerase-encoding genes by complementation of an Escherichia coli polA mutant. Nine novel genes encoding complete DNA polymerase I proteins or domains typical of these proteins were recovered.

scholarly journals Synthetic Nucleotides as Probes of DNA Polymerase Specificity

2012 ◽  
Vol 2012 ◽  
pp. 1-17 ◽  
Author(s):  
Jason M. Walsh ◽  
Penny J. Beuning
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerases ◽  
Polymerase Activity ◽  
Chemical Diversity ◽  
Dna Polymerase I ◽  
Mutagenic Potential ◽  
Natural Agents ◽  
Polymerase I ◽  
Y Family ◽  
Insight Into

The genetic code is continuously expanding with new nucleobases designed to suit specific research needs. These synthetic nucleotides are used to study DNA polymerase dynamics and specificity and may even inhibit DNA polymerase activity. The availability of an increasing chemical diversity of nucleotides allows questions of utilization by different DNA polymerases to be addressed. Much of the work in this area deals with the A family DNA polymerases, for example,Escherichia coliDNA polymerase I, which are DNA polymerases involved in replication and whose fidelity is relatively high, but more recent work includes other families of polymerases, including the Y family, whose members are known to be error prone. This paper focuses on the ability of DNA polymerases to utilize nonnatural nucleotides in DNA templates or as the incoming nucleoside triphosphates. Beyond the utility of nonnatural nucleotides as probes of DNA polymerase specificity, such entities can also provide insight into the functions of DNA polymerases when encountering DNA that is damaged by natural agents. Thus, synthetic nucleotides provide insight into how polymerases deal with nonnatural nucleotides as well as into the mutagenic potential of nonnatural nucleotides.

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