Protein-primed DNA replication: role of inverted terminal repeats in the Escherichia coli bacteriophage PRD1 life cycle.

1993 ◽  
Vol 67 (8) ◽  
pp. 4696-4703 ◽  
Author(s):  
H Savilahti ◽  
D H Bamford
Keyword(s):  
Escherichia Coli ◽  
Life Cycle ◽  
Dna Replication ◽  
Bacteriophage Prd1 ◽  
Inverted Terminal Repeats ◽  
Terminal Repeats

scholarly journals Role of DNA Replication and Repair in Thymineless Death in Escherichia coli

2006 ◽  
Vol 188 (14) ◽  
pp. 5286-5288 ◽  
Author(s):  
Pamela A. Morganroth ◽  
Philip C. Hanawalt
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Excision Repair ◽  
Thymine Starvation ◽  
Nucleotide Excision ◽  
Dna Replication And Repair ◽  
Thymineless Death ◽  
Dna Repair Mutants ◽  
Rule Out

ABSTRACT Inhibition of DNA replication with hydroxyurea during thymine starvation of Escherichia coli shows that active DNA synthesis is not required for thymineless death (TLD). Hydroxyurea experiments and thymine starvation of lexA3 and uvrA DNA repair mutants rule out unbalanced growth, the SOS response, and nucleotide excision repair as explanations for TLD.

scholarly journals The Role of Replication Clamp-Loader Protein HolC of Escherichia coli in Overcoming Replication/Transcription Conflicts

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Deani L. Cooper ◽  
Taku Harada ◽  
Samia Tamazi ◽  
Alexander E. Ferrazzoli ◽  
Susan T. Lovett
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Rna Polymerase ◽  
Transcription Elongation ◽  
Clamp Loader ◽  
Content Type ◽  
Growth Defects ◽  
Transcriptional Termination ◽  
Transcription Complexes

ABSTRACT In Escherichia coli, DNA replication is catalyzed by an assembly of proteins, the DNA polymerase III holoenzyme. This complex includes the polymerase and proofreading subunits, the processivity clamp, and clamp loader complex. The holC gene encodes an accessory protein (known as χ) to the core clamp loader complex and is the only protein of the holoenzyme that binds to single-strand DNA binding protein, SSB. HolC is not essential for viability, although mutants show growth impairment, genetic instability, and sensitivity to DNA damaging agents. In this study, we isolate spontaneous suppressor mutants in a ΔholC strain and identify these by whole-genome sequencing. Some suppressors are alleles of RNA polymerase, suggesting that transcription is problematic for holC mutant strains, or alleles of sspA, encoding stringent starvation protein. Using a conditional holC plasmid, we examine factors affecting transcription elongation and termination for synergistic or suppressive effects on holC mutant phenotypes. Alleles of RpoA (α), RpoB (β), and RpoC (β′) RNA polymerase holoenzyme can partially suppress loss of HolC. In contrast, mutations in transcription factors DksA and NusA enhanced the inviability of holC mutants. HolC mutants showed enhanced sensitivity to bicyclomycin, a specific inhibitor of Rho-dependent termination. Bicyclomycin also reverses suppression of holC by rpoA, rpoC, and sspA. An inversion of the highly expressed rrnA operon exacerbates the growth defects of holC mutants. We propose that transcription complexes block replication in holC mutants and that Rho-dependent transcriptional termination and DksA function are particularly important to sustain viability and chromosome integrity. IMPORTANCE Transcription elongation complexes present an impediment to DNA replication. We provide evidence that one component of the replication clamp loader complex, HolC, of Escherichia coli is required to overcome these blocks. This genetic study of transcription factor effects on holC growth defects implicates Rho-dependent transcriptional termination and DksA function as critical. It also implicates, for the first time, a role of SspA, stringent starvation protein, in avoidance or tolerance of replication/replication conflicts. We speculate that HolC helps avoid or resolve collisions between replication and transcription complexes, which become toxic in HolC’s absence.

scholarly journals The Deacetylase SIRT1 Regulates the Replication Properties of Human Papillomavirus 16 E1 and E2

2017 ◽  
Vol 91 (10) ◽  
Author(s):  
Dipon Das ◽  
Nathan Smith ◽  
Xu Wang ◽  
Iain M. Morgan
Keyword(s):  
Life Cycle ◽  
Dna Replication ◽  
Viral Replication ◽  
Viral Proteins ◽  
Viral Life Cycle ◽  
Human Papillomaviruses ◽  
Dependent Manner ◽  
Viral Origin ◽  
Replication Complex

ABSTRACT Human papillomaviruses (HPV) replicate their genomes in differentiating epithelium using the viral proteins E1 and E2 in association with host proteins. While the roles of E1 and E2 in this process are understood, the host factors involved and how they interact with and regulate E1-E2 are not. Our previous work identified the host replication and repair factor TopBP1 as an E2 partner protein essential for optimal E1-E2 replication and for the viral life cycle. The role of TopBP1 in host DNA replication is regulated by the class III deacetylase SIRT1; activation of the DNA damage response prevents SIRT1 deacetylation of TopBP1, resulting in a switch from DNA replication to repair functions for this protein and cell cycle arrest. Others have demonstrated an essential role for SIRT1 in regulation of the HPV31 life cycle; here, we report that SIRT1 can directly regulate HPV16 E1-E2-mediated DNA replication. SIRT1 is part of the E1-E2 DNA replication complex and is recruited to the viral origin of replication in an E1-E2-dependent manner. CRISPR/Cas9 was used to generate C33a clones with undetectable SIRT1 expression and lack of SIRT1 elevated E1-E2 DNA replication, in part due to increased acetylation and stabilization of the E2 protein in the absence of SIRT1. The results demonstrate that SIRT1 is a member of, and can regulate, the HPV16 replication complex. We discuss the potential role of this protein in the viral life cycle. IMPORTANCE HPV are causative agents in a number of human diseases, and currently only the symptoms of these diseases are treated. To identify novel therapeutic approaches for combating these diseases, the viral life cycle must be understood in more detail. This report demonstrates that a cellular enzyme, SIRT1, is part of the HPV16 DNA replication complex and is brought to the viral genome by the viral proteins E1 and E2. Using gene editing technology (CRISPR/Cas9), the SIRT1 gene was removed from cervical cancer cells. The consequence of this was that viral replication was elevated, probably due to a stabilization of the viral replication factor E2. The overall results demonstrate that an enzyme with known inhibitors, SIRT1, plays an important role in controlling how HPV16 makes copies of itself. Targeting this enzyme could be a new therapeutic approach for combating HPV spread and disease.

scholarly journals The role of replication clamp-loader protein HolC of Escherichia coli in overcoming replication / transcription conflicts

2020 ◽  
Author(s):  
Deani L. Cooper ◽  
Taku Harada ◽  
Samia Tamazi ◽  
Alexander E. Ferrazzoli ◽  
Susan T. Lovett
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Rna Polymerase ◽  
Transcription Elongation ◽  
Specific Inhibitor ◽  
Clamp Loader ◽  
Enhanced Sensitivity ◽  
Transcriptional Termination ◽  
Transcription Complexes

ABSTRACTIn Escherichia coli, DNA replication is catalyzed by an assembly of proteins, the DNA polymerase III holoenzyme. This complex includes the polymerase and proofreading subunits as well as the processivity clamp and clamp loader complex. The holC gene encodes an accessory protein (known as x) to the core clamp loader complex and is the only protein of the holoenzyme that binds to single-strand DNA binding protein, SSB. HolC is not essential for viability although mutants show growth impairment, genetic instability and sensitivity to DNA damaging agents. In this study, to elucidate the role of HolC in replication, we isolate spontaneous suppressor mutants in a holCΔ strain and identify these by whole genome sequencing. Some suppressors are alleles of RNA polymerase, suggesting that transcription is problematic for holC mutant strains or sspA, stringent starvation protein. Using a conditional holC plasmid, we examine factors affecting transcription elongation and termination for synergistic or suppressive effects on holC mutant phenotypes. Alleles of RpoA (α), RpoB (β) and RpoC (β’) RNA polymerase holoenzyme can partially suppress loss of HolC. In contrast, mutations in transcription factors DksA and NusA enhanced the inviability of holC mutants. Mfd had no effect nor did elongation factors GreA and GreB. HolC mutants showed enhanced sensitivity to bicyclomycin, a specific inhibitor of Rho-dependent termination. Bicyclomycin also reverses suppression of holC by rpoA rpoC and sspA.These results are consistent with the hypothesis that transcription complexes block replication in holC mutants and Rho-dependent transcriptional termination and DksA function are particularly important to sustain viability and chromosome integrity.IMPORTANCETranscription elongation complexes present an impediment to DNA replication. We provide evidence that one component of the replication clamp loader complex, HolC, of E. coli is required to overcome these blocks. This genetic study of transcription factor effects on holC growth defects implicates Rho-dependent transcriptional termination and DksA function as critical. It also implicates, for the first time, a role of SspA, stringent starvation protein, in avoidance or tolerance of replication/replication conflicts. We speculate that HolC helps resolve codirectional collisions between replication and transcription complexes, which become toxic in HolC’s absence.

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