DNA polymerase III protein, HolC, helps resolve replication/transcription conflicts

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 holC∆ strain and identify these by whole genome sequencing. Some suppressors are alleles of RNA polymerase, suggesting that transcription is problematic for holC mutant strains, and of 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. 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 Rho-dependent transcriptional termination and DksA function are particularly important to sustain viability and chromosome integrity.

Download Full-text

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

10.1101/2020.12.02.408393 ◽

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.

Download Full-text

Faculty Opinions recommendation of Mechanism of processivity clamp opening by the delta subunit wrench of the clamp loader complex of E. coli DNA polymerase III.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1001104.10052 ◽

2001 ◽

Author(s):

Janet Lindsley

Keyword(s):

Dna Polymerase ◽

Dna Polymerase Iii ◽

Clamp Loader ◽

E Coli ◽

Polymerase Iii ◽

Delta Subunit

Download Full-text

Long-Range PCR Amplification of DNA by DNA Polymerase III Holoenzyme from Thermus thermophilus

Enzyme Research ◽

10.1155/2015/837842 ◽

2015 ◽

Vol 2015 ◽

pp. 1-16 ◽

Cited By ~ 4

Author(s):

Wendy Ribble ◽

Shawn D. Kane ◽

James M. Bullard

Keyword(s):

Dna Synthesis ◽

Dna Polymerase ◽

Thermus Thermophilus ◽

Buffer System ◽

Dna Polymerase Iii ◽

Clamp Loader ◽

Native Form ◽

Polymerase Iii ◽

Essential Components ◽

Pol Iii

DNA replication in bacteria is accomplished by a multicomponent replicase, the DNA polymerase III holoenzyme (pol III HE). The three essential components of the pol III HE are the α polymerase, the β sliding clamp processivity factor, and the DnaX clamp-loader complex. We report here the assembly of the functional holoenzyme from Thermus thermophilus (Tth), an extreme thermophile. The minimal holoenzyme capable of DNA synthesis consists of α, β and DnaX (τ and γ), δ and δ′ components of the clamp-loader complex. The proteins were each cloned and expressed in a native form. Each component of the system was purified extensively. The minimum holoenzyme from these five purified subunits reassembled is sufficient for rapid and processive DNA synthesis. In an isolated form the α polymerase was found to be unstable at temperatures above 65°C. We were able to increase the thermostability of the pol III HE to 98°C by addition and optimization of various buffers and cosolvents. In the optimized buffer system we show that a replicative polymerase apparatus, Tth pol III HE, is capable of rapid amplification of regions of DNA up to 15,000 base pairs in PCR reactions.

Download Full-text

Solution study of the Escherichia coli DNA polymerase III clamp loader reveals the location of the dynamic ψχ heterodimer

Structural Dynamics ◽

10.1063/1.4927407 ◽

2015 ◽

Vol 2 (5) ◽

pp. 054701 ◽

Cited By ~ 3

Author(s):

Farzaneh Tondnevis ◽

Richard E. Gillilan ◽

Linda B. Bloom ◽

Robert McKenna

Keyword(s):

Escherichia Coli ◽

Dna Polymerase ◽

Dna Polymerase Iii ◽

Clamp Loader ◽

Solution Study ◽

Polymerase Iii

Download Full-text

Mechanism of Processivity Clamp Opening by the Delta Subunit Wrench of the Clamp Loader Complex of E. coli DNA Polymerase III

Cell ◽

10.1016/s0092-8674(01)00462-7 ◽

2001 ◽

Vol 106 (4) ◽

pp. 417-428 ◽

Cited By ~ 183

Author(s):

David Jeruzalmi ◽

Olga Yurieva ◽

Yanxiang Zhao ◽

Matthew Young ◽

Jelena Stewart ◽

...

Keyword(s):

Dna Polymerase ◽

Dna Polymerase Iii ◽

Clamp Loader ◽

E Coli ◽

Polymerase Iii ◽

Delta Subunit

Download Full-text

Escherichia coli mismatch repair protein MutL interacts with the clamp loader subunits of DNA polymerase III

Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis ◽

10.1016/j.mrfmmm.2007.07.008 ◽

2008 ◽

Vol 637 (1-2) ◽

pp. 101-110 ◽

Cited By ~ 18

Author(s):

Feng Li ◽

Qin Liu ◽

Yuan-Yuan Chen ◽

Zi-Niu Yu ◽

Zhi-Ping Zhang ◽

...

Keyword(s):

Escherichia Coli ◽

Mismatch Repair ◽

Dna Polymerase ◽

Dna Polymerase Iii ◽

Clamp Loader ◽

Polymerase Iii ◽

Repair Protein ◽

Mismatch Repair Protein

Download Full-text

Protein trafficking on sliding clamps

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2003.1361 ◽

2004 ◽

Vol 359 (1441) ◽

pp. 25-30 ◽

Cited By ~ 11

Author(s):

Francisco López de Saro ◽

Roxana E. Georgescu ◽

Frank Leu ◽

Mike O'Donnell

Keyword(s):

Escherichia Coli ◽

Traffic Flow ◽

Dna Polymerase ◽

Protein Trafficking ◽

Dna Structure ◽

Dna Polymerase Iii ◽

Clamp Loader ◽

Polymerase Iii ◽

Sliding Clamps

The sliding clamps of chromosomal replicases are acted upon by both the clamp loader and DNA polymerase. Several other proteins and polymerases also interact with the clamp. These proteins bind the clamp at the same spot and use it in sequential fashion. First the clamp loader must bind the clamp in order to load it onto DNA, but directly thereafter the clamp loader must clear away from the clamp so it can be used by the replicative DNA polymerase. At the end of replication, the replicase is ejected from the clamp, which presumably allows the clamp to interact with yet other proteins after its use by the replicase. This paper describes how different proteins in the Escherichia coli replicase, DNA polymerase III holoenzyme, coordinate their traffic flow on the clamp. The mechanism by which traffic flow on the β; clamp is directed is based on competition of the proteins for the clamp, where DNA structure modulates the competition. It seems likely that the principles will generalize to a traffic flow of other factors on these circular clamp proteins.

Download Full-text