Effects of Benzo[a]pyrene Adduct Stereochemistry on Downstream DNA Replication in Vitro:  Evidence for Different Adduct Conformations within the Active Site of DNA Polymerase I (Klenow Fragment)†

Biochemistry ◽  
2002 ◽  
Vol 41 (13) ◽  
pp. 4467-4479 ◽  
Author(s):  
Yuriy O. Alekseyev ◽  
Louis J. Romano
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Active Site ◽  
Klenow Fragment ◽  
Dna Polymerase I ◽  
Polymerase I

Mechanism of DNA replication fidelity for three mutants of DNA polymerase I: Klenow fragment KF(exo+), KF(polA5), and KF(exo-)

Biochemistry ◽  
1991 ◽  
Vol 30 (5) ◽  
pp. 1441-1448 ◽  
Author(s):  
Bryan T. Eger ◽  
Robert D. Kuchta ◽  
Steven S. Carroll ◽  
Patricia A. Benkovic ◽  
Michael E. Dahlberg ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Klenow Fragment ◽  
Dna Polymerase I ◽  
Replication Fidelity ◽  
Polymerase I ◽  
Dna Replication Fidelity

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.

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 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.

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.

scholarly journals The mechanism of recA polA lethality: suppression by RecA-independent recombination repair activated by the lexA(Def) mutation in Escherichia coli.

Genetics ◽  
1995 ◽  
Vol 139 (4) ◽  
pp. 1483-1494 ◽  
Author(s):  
Y Cao ◽  
T Kogoma
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Reca Protein ◽  
Minor Role ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Dna Polymerase I ◽  
Recombination Repair ◽  
Polymerase I

Abstract The mechanism of recA polA lethality in Escherichia coli has been studied. Complementation tests have indicated that both the 5'-->3' exonuclease and the polymerization activities of DNA polymerase I are essential for viability in the absence of RecA protein, whereas the viability and DNA replication of DNA polymerase I-defective cells depend on the recombinase activity of RecA. An alkaline sucrose gradient sedimentation analysis has indicated that RecA has only a minor role in Okazaki fragment processing. Double-strand break repair is proposed for the major role of RecA in the absence of DNA polymerase I. The lexA(Def)::Tn5 mutation has previously been shown to suppress the temperature-sensitive growth of recA200(Ts) polA25::spc mutants. The lexA(Def) mutation can alleviate impaired DNA synthesis in the recA200(Ts) polA25::spc mutant cells at the restrictive temperature. recF+ is essential for this suppression pathway. recJ and recQ mutations have minor but significant adverse effects on the suppression. The recA200(Ts) allele in the recA200(Ts) polA25::spc lexA(Def) mutant can be replaced by delta recA, indicating that the lexA(Def)-induced suppression is RecA independent. lexA(Def) reduces the sensitivity of delta recA polA25::spc cells to UV damage by approximately 10(4)-fold. lexA(Def) also restores P1 transduction proficiency to the delta recA polA25::spc mutant to a level that is 7.3% of the recA+ wild type. These results suggest that lexA(Def) activates a RecA-independent, RecF-dependent recombination repair pathway that suppresses the defect in DNA replication in recA polA double mutants.

Sign in / Sign up

Export Citation Format

Share Document