scholarly journals Effect of dNTP pool alterations on fidelity of leading and lagging strand DNA replication in E. coli

2014 ◽  
Vol 759 ◽  
pp. 22-28 ◽  
Author(s):  
Damian Gawel ◽  
Iwona J. Fijalkowska ◽  
Piotr Jonczyk ◽  
Roel M. Schaaper
Keyword(s):  
Dna Replication ◽  
Dntp Pool ◽  
E Coli ◽  
Lagging Strand

scholarly journals Near-continuously synthesized leading strands inEscherichia coliare broken by ribonucleotide excision

2019 ◽  
Vol 116 (4) ◽  
pp. 1251-1260 ◽  
Author(s):  
Glen E. Cronan ◽  
Elena A. Kouzminova ◽  
Andrei Kuzminov
Keyword(s):  
Dna Replication ◽  
Excision Repair ◽  
Chromosomal Dna ◽  
E Coli ◽  
Okazaki Fragments ◽  
Leading Strand ◽  
Replication Intermediates ◽  
Lagging Strand

In vitro, purified replisomes drive model replication forks to synthesize continuous leading strands, even without ligase, supporting the semidiscontinuous model of DNA replication. However, nascent replication intermediates isolated from ligase-deficientEscherichia colicomprise only short (on average 1.2-kb) Okazaki fragments. It was long suspected that cells replicate their chromosomal DNA by the semidiscontinuous mode observed in vitro but that, in vivo, the nascent leading strand was artifactually fragmented postsynthesis by excision repair. Here, using high-resolution separation of pulse-labeled replication intermediates coupled with strand-specific hybridization, we show that excision-proficientE. coligenerates leading-strand intermediates >10-fold longer than lagging-strand Okazaki fragments. Inactivation of DNA-repair activities, including ribonucleotide excision, further increased nascent leading-strand size to ∼80 kb, while lagging-strand Okazaki fragments remained unaffected. We conclude that in vivo, repriming occurs ∼70× less frequently on the leading versus lagging strands, and that DNA replication inE. coliis effectively semidiscontinuous.

scholarly journals Staphylococcus aureus Helicase but Not Escherichia coli Helicase Stimulates S. aureus Primase Activity and Maintains Initiation Specificity

2006 ◽  
Vol 188 (13) ◽  
pp. 4673-4680 ◽  
Author(s):  
Scott A. Koepsell ◽  
Marilynn A. Larson ◽  
Mark A. Griep ◽  
Steven H. Hinrichs
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Dna Replication ◽  
Full Length ◽  
Recognition Sequence ◽  
Dna Templates ◽  
E Coli ◽  
The Third ◽  
Leading Strand ◽  
Lagging Strand

ABSTRACT Bacterial primases are essential for DNA replication due to their role in polymerizing the formation of short RNA primers repeatedly on the lagging-strand template and at least once on the leading-strand template. The ability of recombinant Staphylococcus aureus DnaG primase to utilize different single-stranded DNA templates was tested using oligonucleotides of the sequence 5′-CAGA (CA)5 XYZ (CA)3-3′, where XYZ represented the variable trinucleotide. These experiments demonstrated that S. aureus primase synthesized RNA primers predominately on templates containing 5′-d(CTA)-3′ or TTA and to a much lesser degree on GTA-containing templates, in contrast to results seen with the Escherichia coli DnaG primase recognition sequence 5′-d(CTG)-3′. Primer synthesis was initiated complementarily to the middle nucleotide of the recognition sequence, while the third nucleotide, an adenosine, was required to support primer synthesis but was not copied into the RNA primer. The replicative helicases from both S. aureus and E. coli were tested for their ability to stimulate either S. aureus or E. coli primase. Results showed that each bacterial helicase could only stimulate the cognate bacterial primase. In addition, S. aureus helicase stimulated the production of full-length primers, whereas E. coli helicase increased the synthesis of only short RNA polymers. These studies identified important differences between E. coli and S. aureus related to DNA replication and suggest that each bacterial primase and helicase may have adapted unique properties optimized for replication.

Replication of Bacteriophage 186 DNA

1972 ◽  
Vol 30 ◽  
pp. 194-195
Author(s):  
Dhruba K. Chattoraj ◽  
Ross B. Inman
Keyword(s):  
Electron Microscopy ◽  
Dna Replication ◽  
Frame Of Reference ◽  
Dna Molecules ◽  
E Coli ◽  
Phage Dna ◽  
Partial Denaturation

Electron microscopy of replicating intermediates has been quite useful in understanding the mechanism of DNA replication in DNA molecules of bacteriophage, mitochondria and plasmids. The use of partial denaturation mapping has made the tool more powerful by providing a frame of reference by which the position of the replicating forks in bacteriophage DNA can be determined on the circular replicating molecules. This provided an easy means to find the origin and direction of replication in λ and P2 phage DNA molecules. DNA of temperate E. coli phage 186 was found to have an unique denaturation map and encouraged us to look into its mode of replication.

scholarly journals E. coli primase and DNA polymerase III holoenzyme are able to bind concurrently to a primed template during DNA replication

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Andrea Bogutzki ◽  
Natalie Naue ◽  
Lidia Litz ◽  
Andreas Pich ◽  
Ute Curth
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Protein Interactions ◽  
Dna Polymerase Iii ◽  
Protein Protein Interactions ◽  
Single Stranded Dna ◽  
E Coli ◽  
Polymerase Iii ◽  
C Terminus ◽  
Pol Iii

Abstract During DNA replication in E. coli, a switch between DnaG primase and DNA polymerase III holoenzyme (pol III) activities has to occur every time when the synthesis of a new Okazaki fragment starts. As both primase and the χ subunit of pol III interact with the highly conserved C-terminus of single-stranded DNA-binding protein (SSB), it had been proposed that the binding of both proteins to SSB is mutually exclusive. Using a replication system containing the origin of replication of the single-stranded DNA phage G4 (G4ori) saturated with SSB, we tested whether DnaG and pol III can bind concurrently to the primed template. We found that the addition of pol III does not lead to a displacement of primase, but to the formation of higher complexes. Even pol III-mediated primer elongation by one or several DNA nucleotides does not result in the dissociation of DnaG. About 10 nucleotides have to be added in order to displace one of the two primase molecules bound to SSB-saturated G4ori. The concurrent binding of primase and pol III is highly plausible, since even the SSB tetramer situated directly next to the 3′-terminus of the primer provides four C-termini for protein-protein interactions.

scholarly journals 3′ → 5′ Exonucleases of DNA Polymerases ϵ and δ Correct Base Analog Induced DNA Replication Errors on Opposite DNA Strands in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (3) ◽  
pp. 717-726 ◽  
Author(s):  
Polina V Shcherbakova ◽  
Youri I Pavlov
Keyword(s):  
Dna Replication ◽  
Dna Polymerases ◽  
Replication Errors ◽  
Dna Strands ◽  
Base Analog ◽  
Chromosome Iii ◽  
Dna Strand ◽  
Dna Replication Errors ◽  
Yeast Mutants ◽  
Lagging Strand

Abstract The base analog 6-N-hydroxylaminopurine (HAP) induces bidirectional GC → AT and AT → GC transitions that are enhanced in DNA polymerase ϵ and δ 3′ → 5′ exonuclease-deficient yeast mutants, pol2-4 and pol3-01, respectively. We have constructed a set of isogenic strains to determine whether the DNA polymerases δ and ϵ contribute equally to proofreading of replication errors provoked by HAP during leading and lagging strand DNA synthesis. Site-specific GC → AT and AT → GC transitions in a Pol→, pol2-4 or pol3-01 genetic background were scored as reversions of ura3 missense alleles. At each site, reversion was increased in only one proofreading-deficient mutant, either pol2-4 or pol3-01, depending on the DNA strand in which HAP incorporation presumably occurred. Measurement of the HAP-induced reversion frequency of the ura3 alleles placed into chromosome III near to the defined active replication origin ARS306 in two orientations indicated that DNA polymerases ϵ and δ correct HAP-induced DNA replication errors on opposite DNA strands.

scholarly journals Dissecting the Control Mechanisms for DNA Replication and Cell Division in E. coli

Cell Reports ◽  
2018 ◽  
Vol 25 (3) ◽  
pp. 761-771.e4 ◽  
Author(s):  
Gabriele Micali ◽  
Jacopo Grilli ◽  
Jacopo Marchi ◽  
Matteo Osella ◽  
Marco Cosentino Lagomarsino
Keyword(s):  
Cell Division ◽  
Dna Replication ◽  
Control Mechanisms ◽  
E Coli

scholarly journals In vitro Assays for Eukaryotic Leading/Lagging Strand DNA Replication

BIO-PROTOCOL ◽  
2017 ◽  
Vol 7 (18) ◽  
Author(s):  
Grant Schauer ◽  
Jeff Finkelstein ◽  
Mike O’Donnell
Keyword(s):  
Dna Replication ◽  
In Vitro Assays ◽  
Lagging Strand

scholarly journals UV- and MMS-induced mutagenesis of lambdaO(am)8 phage under nonpermissive conditions for phage DNA replication.

2003 ◽  
Vol 50 (4) ◽  
pp. 921-939 ◽  
Author(s):  
Joanna Krwawicz ◽  
Anna Czajkowska ◽  
Magdalena Felczak ◽  
Irena Pietrzykowska
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Excision Repair ◽  
Protein S ◽  
Dna Lesions ◽  
E Coli ◽  
Phage Dna ◽  
Induced Mutagenesis ◽  
C Proteins ◽  
Inducible Protein

Mutagenesis in Escherichia coli, a subject of many years of study is considered to be related to DNA replication. DNA lesions nonrepaired by the error-free nucleotide excision repair (NER), base excision repair (BER) and recombination repair (RR), stop replication at the fork. Reinitiation needs translesion synthesis (TLS) by DNA polymerase V (UmuC), which in the presence of accessory proteins, UmuD', RecA and ssDNA-binding protein (SSB), has an ability to bypass the lesion with high mutagenicity. This enables reinitiation and extension of DNA replication by DNA polymerase III (Pol III). We studied UV- and MMS-induced mutagenesis of lambdaO(am)8 phage in E. coli 594 sup+ host, unable to replicate the phage DNA, as a possible model for mutagenesis induced in nondividing cells (e.g. somatic cells). We show that in E. coli 594 sup+ cells UV- and MMS-induced mutagenesis of lambdaO(am)8 phage may occur. This mutagenic process requires both the UmuD' and C proteins, albeit a high level of UmuD' and low level of UmuC seem to be necessary and sufficient. We compared UV-induced mutagenesis of lambdaO(am)8 in nonpermissive (594 sup+) and permissive (C600 supE) conditions for phage DNA replication. It appeared that while the mutagenesis of lambdaO(am)8 in 594 sup+ requires the UmuD' and C proteins, which can not be replaced by other SOS-inducible protein(s), in C600 supE their functions may be replaced by other inducible protein(s), possibly DNA polymerase IV (DinB). Mutations induced under nonpermissive conditions for phage DNA replication are resistant to mismatch repair (MMR), while among those induced under permissive conditions, only about 40% are resistant.

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