scholarly journals Topological stress is responsible for the detrimental outcomes of head-on replication-transcription conflicts

2019 ◽  
Author(s):  
Kevin S. Lang ◽  
Houra Merrikh
Keyword(s):  
Conflict Resolution ◽  
Rna Polymerase ◽  
Genome Organization ◽  
Dna Gyrase ◽  
Fundamental Difference ◽  
Loop Formation ◽  
Chromosome Duplication ◽  
Leading Strand ◽  
Lagging Strand ◽  
Negative Supercoils

AbstractConflicts between the replication and transcription machineries have profound effects on chromosome duplication, genome organization, as well as evolution across species. Head-on conflicts (lagging strand genes) are significantly more detrimental than co-directional conflicts (leading strand genes). The source of this fundamental difference is unknown. Here, we report that topological stress underlies this difference. We find that head-on conflict resolution requires the relaxation of positive supercoils DNA gyrase and Topo IV. Interestingly, we find that after positive supercoil resolution, gyrase introduces excessive negative supercoils at head-on conflict regions, driving pervasive R-loop formation. The formation of these R-Loops through gyrase activity is most likely caused by the diffusion of negative supercoils through RNA polymerase spinning. Altogether, our results address a longstanding question regarding replication-transcription conflicts by revealing the fundamental mechanistic difference between the two types of encounters.

scholarly journals RNA polymerase II plays an active role in the formation of gene loops through the Rpb4 subunit

2019 ◽  
Vol 47 (17) ◽  
pp. 8975-8987 ◽  
Author(s):  
Paula Allepuz-Fuster ◽  
Michael J O’Brien ◽  
Noelia González-Polo ◽  
Bianca Pereira ◽  
Zuzer Dhoondia ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Essential Role ◽  
Transcription Termination ◽  
Active Role ◽  
The Other ◽  
Loop Formation ◽  
Direct Role

AbstractGene loops are formed by the interaction of initiation and termination factors occupying the distal ends of a gene during transcription. RNAPII is believed to affect gene looping indirectly owing to its essential role in transcription. The results presented here, however, demonstrate a direct role of RNAPII in gene looping through the Rpb4 subunit. 3C analysis revealed that gene looping is abolished in the rpb4Δ mutant. In contrast to the other looping-defective mutants, rpb4Δ cells do not exhibit a transcription termination defect. RPB4 overexpression, however, rescued the transcription termination and gene looping defect of sua7-1, a mutant of TFIIB. Furthermore, RPB4 overexpression rescued the ssu72-2 gene looping defect, while SSU72 overexpression restored the formation of gene loops in rpb4Δ cells. Interestingly, the interaction of TFIIB with Ssu72 is compromised in rpb4Δ cells. These results suggest that the TFIIB–Ssu72 interaction, which is critical for gene loop formation, is facilitated by Rpb4. We propose that Rpb4 is promoting the transfer of RNAPII from the terminator to the promoter for reinitiation of transcription through TFIIB–Ssu72 mediated gene looping.

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 In Vitro Evaluation of CBR-2092, a Novel Rifamycin-Quinolone Hybrid Antibiotic: Studies of the Mode of Action in Staphylococcus aureus

2008 ◽  
Vol 52 (7) ◽  
pp. 2313-2323 ◽  
Author(s):  
Gregory T. Robertson ◽  
Eric J. Bonventre ◽  
Timothy B. Doyle ◽  
Qun Du ◽  
Leonard Duncan ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Rna Polymerase ◽  
Mode Of Action ◽  
Bacterial Infections ◽  
Dna Gyrase ◽  
Topoisomerase Iv ◽  
Dna Topoisomerase ◽  
Resistant Variant ◽  
Proven Efficacy

ABSTRACT Rifamycins have proven efficacy in the treatment of persistent bacterial infections. However, the frequency with which bacteria develop resistance to rifamycin agents restricts their clinical use to antibiotic combination regimens. In a program directed toward the synthesis of rifamycins with a lower propensity to elicit resistance development, a series of compounds were prepared that covalently combine rifamycin and quinolone pharmacophores to form stable hybrid antibacterial agents. We describe mode-of-action studies with Staphylococcus aureus of CBR-2092, a novel hybrid that combines the rifamycin SV and 4H-4-oxo-quinolizine pharmacophores. In biochemical studies, CBR-2092 exhibited rifampin-like potency as an inhibitor of RNA polymerase, was an equipotent (balanced) inhibitor of DNA gyrase and DNA topoisomerase IV, and retained activity against a prevalent quinolone-resistant variant. Macromolecular biosynthesis studies confirmed that CBR-2092 has rifampin-like effects on RNA synthesis in rifampin-susceptible strains and quinolone-like effects on DNA synthesis in rifampin-resistant strains. Studies of mutant strains that exhibited reduced susceptibility to CBR-2092 further substantiated RNA polymerase as the primary cellular target of CBR-2092, with DNA gyrase and DNA topoisomerase IV being secondary and tertiary targets, respectively, in strains exhibiting preexisting rifampin resistance. In contrast to quinolone comparator agents, no strains with altered susceptibility to CBR-2092 were found to exhibit changes consistent with altered efflux properties. The combined data indicate that CBR-2092 may have potential utility in monotherapy for the treatment of persistent S. aureus infections.

scholarly journals Primer release is the rate-limiting event in lagging-strand synthesis mediated by the T7 replisome

2016 ◽  
Vol 113 (21) ◽  
pp. 5916-5921 ◽  
Author(s):  
Alfredo J. Hernandez ◽  
Seung-Joo Lee ◽  
Charles C. Richardson
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Fragment Length ◽  
Dna Binding Protein ◽  
Okazaki Fragment ◽  
Dna Primase ◽  
Dna Strands ◽  
Leading Strand ◽  
Rate Limiting ◽  
Lagging Strand

DNA replication occurs semidiscontinuously due to the antiparallel DNA strands and polarity of enzymatic DNA synthesis. Although the leading strand is synthesized continuously, the lagging strand is synthesized in small segments designated Okazaki fragments. Lagging-strand synthesis is a complex event requiring repeated cycles of RNA primer synthesis, transfer to the lagging-strand polymerase, and extension effected by cooperation between DNA primase and the lagging-strand polymerase. We examined events controlling Okazaki fragment initiation using the bacteriophage T7 replication system. Primer utilization by T7 DNA polymerase is slower than primer formation. Slow primer release from DNA primase allows the polymerase to engage the complex and is followed by a slow primer handoff step. The T7 single-stranded DNA binding protein increases primer formation and extension efficiency but promotes limited rounds of primer extension. We present a model describing Okazaki fragment initiation, the regulation of fragment length, and their implications for coordinated leading- and lagging-strand DNA synthesis.

scholarly journals Molecular Basis for the Differential Quinolone Susceptibility of Mycobacterial DNA Gyrase

2014 ◽  
Vol 58 (4) ◽  
pp. 2013-2020 ◽  
Author(s):  
Rupesh Kumar ◽  
Bhavani Shankar Madhumathi ◽  
Valakunja Nagaraja
Keyword(s):  
Differential Susceptibility ◽  
Dna Gyrase ◽  
Drug Binding ◽  
Double Strand Breaks ◽  
Type Ii ◽  
Content Type ◽  
Strand Breaks ◽  
E Coli ◽  
Dna Complex ◽  
Negative Supercoils

ABSTRACTDNA gyrase is a type II topoisomerase that catalyzes the introduction of negative supercoils in the genomes of eubacteria. Fluoroquinolones (FQs), successful as drugs clinically, target the enzyme to trap the gyrase-DNA complex, leading to the accumulation of double-strand breaks in the genome. Mycobacteria are less susceptible to commonly used FQs. However, an 8-methoxy-substituted FQ, moxifloxacin (MFX), is a potent antimycobacterial, and a higher susceptibility of mycobacterial gyrase to MFX has been demonstrated. Although several models explain the mechanism of FQ action and gyrase-DNA-FQ interaction, the basis for the differential susceptibility of mycobacterial gyrase to various FQs is not understood. We have addressed the basis of the differential susceptibility of the gyrase and revisited the mode of action of FQs. We demonstrate that FQs bind bothEscherichia coliandMycobacterium tuberculosisgyrases in the absence of DNA and that the addition of DNA enhances the drug binding. The FQs bind primarily to the GyrA subunit of mycobacterial gyrase, while inE. coliholoenzyme is the target. The binding of MFX to GyrA ofM. tuberculosiscorrelates with its effectiveness as a better inhibitor of the enzyme and its efficacy in cell killing.

scholarly journals Human mismatch repair system corrects errors produced during lagging strand replication more effectively

2016 ◽  
Author(s):  
Maria Andrianova ◽  
Georgii A Bazykin ◽  
Sergey Nikolaev ◽  
Vladimir Seplyarskiy
Keyword(s):  
Mismatch Repair ◽  
Mutation Rates ◽  
Repair System ◽  
Wild Type ◽  
Strand Asymmetry ◽  
Exonuclease Domain ◽  
Mismatch Repair System ◽  
Dna Strands ◽  
Leading Strand ◽  
Lagging Strand

Mismatch repair (MMR) is one of the main systems maintaining fidelity of replication. Different effectiveness in correction of errors produced during replication of the leading and the lagging DNA strands was reported in yeast, but this effect is poorly studied in humans. Here, we use MMR-deficient (MSI) and MMR-proficient (MSS) cancer samples to investigate properties of the human MMR. MSI, but not MSS, cancers demonstrate unequal mutation rates between the leading and the lagging strands. The direction of strand asymmetry in MSI cancers matches that observed in cancers with mutated exonuclease domain of polymerase δ, indicating that polymerase δ contributes more mutations than its leading-strand counterpart, polymerase ε. As polymerase δ primarily synthesizes DNA during the lagging strand replication, this implies that mutations produced in wild type cells during lagging strand replication are repaired by the MMR ~3 times more effectively, compared to those produced on the leading strand.

scholarly journals The CMG helicase bypasses DNA protein cross-links to facilitate their repair

2018 ◽  
Author(s):  
Justin L. Sparks ◽  
Alan O. Gao ◽  
Markus Räschle ◽  
Nicolai B. Larsen ◽  
Matthias Mann ◽  
...  
Keyword(s):  
Replication Fork ◽  
Dna Helicase ◽  
Genome Integrity ◽  
Cross Link ◽  
Replication Fork Progression ◽  
Egg Extracts ◽  
Cross Links ◽  
Leading Strand ◽  
Nucleoprotein Complexes ◽  
Lagging Strand

SummaryCovalent and non-covalent nucleoprotein complexes impede replication fork progression and thereby threaten genome integrity. UsingXenopus laevisegg extracts, we previously showed that when a replication fork encounters a covalent DNA-protein cross-link (DPC) on the leading strand template, the DPC is degraded to a short peptide, allowing its bypass by translesion synthesis polymerases. Strikingly, we show here that when DPC proteolysis is blocked, the replicative DNA helicase (CMG), which travels on the leading strand template, still bypasses the intact DPC. The DNA helicase RTEL1 facilitates bypass, apparently by translocating along the lagging strand template and generating single-stranded DNA downstream of the DPC. Remarkably, RTEL1 is required for efficient DPC proteolysis, suggesting that CMG bypass of a DPC normally precedes its proteolysis. RTEL1 also promotes fork progression past non-covalent protein-DNA complexes. Our data suggest a unified model for the replisome’s response to nucleoprotein barriers.

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