scholarly journals Identification of R/KRRY motif in Klenow Fragment of E. coli DNA polymerase I: Its role in coordinating polymerase and exonuclease activity

2007 ◽  
Vol 21 (5) ◽  
Author(s):  
Pinky Kukreti ◽  
Kamalendra Singh ◽  
Mukund J. Modak
Keyword(s):  
Dna Polymerase ◽  
Exonuclease Activity ◽  
Klenow Fragment ◽  
Dna Polymerase I ◽  
E Coli ◽  
Polymerase I

How E. coli DNA polymerase I (klenow fragment) distinguishes between deoxy- and dideoxynucleotides

1998 ◽  
Vol 278 (1) ◽  
pp. 147-165 ◽  
Author(s):  
Mekbib Astatke ◽  
Nigel D.F Grindley ◽  
Catherine M Joyce
Keyword(s):  
Dna Polymerase ◽  
Klenow Fragment ◽  
Dna Polymerase I ◽  
E Coli ◽  
Polymerase I

Labeling of Synthetic Oligonucleotides Using the Klenow Fragment of E. coli DNA Polymerase I

2021 ◽  
Vol 2021 (10) ◽  
pp. pdb.prot100693
Author(s):  
Michael R. Green ◽  
Joseph Sambrook
Keyword(s):  
Dna Polymerase ◽  
Oligonucleotide Probes ◽  
Klenow Fragment ◽  
Dna Polymerase I ◽  
E Coli ◽  
Specific Activities ◽  
Radiolabeled Probe ◽  
Polymerase I ◽  
Synthetic Oligonucleotides ◽  
Radioactive Atoms

In this method, a short primer is hybridized to an oligonucleotide template whose sequence is the complement of the desired radiolabeled probe. The primer is then extended using the Klenow fragment to incorporate [α-32P]dNTPs in a template-directed manner. After the reaction, the template and product are separated by denaturation followed by electrophoresis through a polyacrylamide gel under denaturing conditions. With this method, it is possible to generate oligonucleotide probes that contain several radioactive atoms per molecule of oligonucleotide and to achieve specific activities as high as 2 × 1010 cpm/µg of probe. Because the end product of the reaction is dsDNA, whose strands must be separated and the labeled product isolated, this method is generally not used to prepare nonradiolabeled oligonucleotides.

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.

Recognition of the primers containing different modified nucleotide units by the Klenow fragment of DNA polymerase I from E coli

Biochimie ◽  
1996 ◽  
Vol 78 (3) ◽  
pp. 201-203
Author(s):  
T.I. Kolocheva ◽  
A.S. Levina ◽  
G.A. Nevinsky
Keyword(s):  
Dna Polymerase ◽  
Klenow Fragment ◽  
Dna Polymerase I ◽  
E Coli ◽  
Polymerase I

scholarly journals The efficiency of interaction of deoxyribonucleosid-5'-mono-, di- and triphosphates with the active centre of E. coli DNA polymerase I Klenow fragment

FEBS Letters ◽  
1989 ◽  
Vol 259 (1) ◽  
pp. 83-85 ◽  
Author(s):  
S.V. Doronin ◽  
G.A. Nevinsky ◽  
T.O. Malygina ◽  
V.N. Podust ◽  
V.V. Khomov ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Klenow Fragment ◽  
Dna Polymerase I ◽  
Active Centre ◽  
E Coli ◽  
Polymerase I

E. coli DNA Polymerase I and the Klenow Fragment

2020 ◽  
Vol 2020 (5) ◽  
pp. pdb.top100743
Author(s):  
Michael R. Green ◽  
Joseph Sambrook
Keyword(s):  
Dna Polymerase ◽  
Klenow Fragment ◽  
Dna Polymerase I ◽  
E Coli ◽  
Polymerase I
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