In Vitro Bypass of Malondialdehyde−Deoxyguanosine Adducts:  Differential Base Selection during Extension by the Klenow Fragment of DNA Polymerase I Is the Critical Determinant of Replication Outcome†

Biochemistry ◽  
2004 ◽  
Vol 43 (37) ◽  
pp. 11828-11835 ◽  
Author(s):  
Muhammed F. Hashim ◽  
James N. Riggins ◽  
Nathalie Schnetz-Boutaud ◽  
Markus Voehler ◽  
Michael P. Stone ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Klenow Fragment ◽  
Dna Polymerase I ◽  
Critical Determinant ◽  
Base Selection ◽  
Polymerase I

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 Crystallization and preliminary X-ray analysis of thePlasmodium falciparumapicoplast DNA polymerase

2015 ◽  
Vol 71 (3) ◽  
pp. 333-337 ◽  
Author(s):  
Morgan E. Milton ◽  
Jun-yong Choe ◽  
Richard B. Honzatko ◽  
Scott W. Nelson
Keyword(s):  
Dna Polymerase ◽  
Structural Information ◽  
Klenow Fragment ◽  
X Ray Diffraction ◽  
Dna Polymerase I ◽  
X Ray ◽  
Cell Parameters ◽  
A Genome ◽  
Reported Data ◽  
Polymerase I

Infection by the parasitePlasmodium falciparumis the leading cause of malaria in humans. The parasite has a unique and essential plastid-like organelle called the apicoplast. The apicoplast contains a genome that undergoes replication and repair through the action of a replicative polymerase (apPOL). apPOL has no direct orthologs in mammalian polymerases and is therefore an attractive antimalarial drug target. No structural information exists for apPOL, and the Klenow fragment ofEscherichia coliDNA polymerase I, which is its closest structural homolog, shares only 28% sequence identity. Here, conditions for the crystallization of and preliminary X-ray diffraction data from crystals ofP. falciparumapPOL are reported. Data complete to 3.5 Å resolution were collected from a single crystal (2 × 2 × 5 µm) using a 5 µm beam. The space groupP6522 (unit-cell parametersa=b= 141.8,c= 149.7 Å, α = β = 90, γ = 120°) was confirmed by molecular replacement. Refinement is in progress.

DNA polymerase I proofreading exonuclease activity is required for endonuclease V repair pathway both in vitro and in vivo

DNA Repair ◽  
2018 ◽  
Vol 64 ◽  
pp. 59-67 ◽  
Author(s):  
Kang-Yi Su ◽  
Liang-In Lin ◽  
Steven D. Goodman ◽  
Rong-Syuan Yen ◽  
Cho-Yuan Wu ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Exonuclease Activity ◽  
Repair Pathway ◽  
Dna Polymerase I ◽  
Endonuclease V ◽  
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
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