scholarly journals Genetic analysis of DinG-family helicase YoaA and its interaction with replication clamp-loader protein HolC in E. coli

2021 ◽  
Author(s):  
Vincent A. Sutera ◽  
Thalia H. Sass ◽  
Scott E. Leonard ◽  
Lingling Wu ◽  
David J. Glass ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Tandem Repeats ◽  
Genomic Stability ◽  
Sos Response ◽  
Template Switching ◽  
Clamp Loader ◽  
E Coli ◽  
C Terminus ◽  
Domains Of Life

The XP-D/DinG family of DNA helicases contributes to genomic stability in all three domains of life. We investigate here the role of one of these proteins, YoaA, of Escherichia coli . In E. coli , YoaA aids tolerance to the nucleoside azidothymidine (AZT), a DNA replication inhibitor and physically interacts with a subunit of the DNA polymerase III holoenzyme, HolC. We map the residues of YoaA required for HolC interaction to its C-terminus by yeast two-hybrid analysis. We propose that this interaction competes with HolC’s interaction with HolD and the rest of the replisome; YoaA indeed inhibits growth when overexpressed, dependent on this interaction region. By gene fusions we show YoaA is repressed by LexA and induced in response to DNA damage as part of the SOS response. Induction of YoaA by AZT is biphasic with an immediate response after treatment and a slower response that peaks in the late log phase of growth. This growth-phase dependent induction by AZT is not blocked by lexA3 (Ind - ), which normally negates its self-cleavage, implying another means to induce the DNA damage response that responds to the nutritional state of the cell. We propose that YoaA helicase activity increases access to the 3’ nascent strand during replication; consistent with this, YoaA appears to aid removal of potential A-to-T transversion mutations in ndk mutants, which are prone to nucleotide misincorporation. We provide evidence that YoaA and its paralog DinG also may initiate template-switching that leads to deletions between tandem repeats in DNA. IMPORTANCE Maintaining genomic stability is crucial for all living organisms. Replication of DNA frequently encounters barriers that must be removed to complete genome duplication. Balancing DNA synthesis with its repair is critical and not entirely understood at a mechanistic level. The YoaA protein, studied here, is required for certain types of DNA repair and interacts in an alternative manner with proteins that catalyze DNA replication. YoaA is part of the well-studied LexA-regulated response to DNA damage, the SOS response. We describe an unusual feature of its regulation that promotes induction after DNA damage as the culture begins to experience starvation. Replication fork repair integrates both DNA damage and nutritional signals. We also show that YoaA affects genomic stability.

scholarly journals Mutational analysis of DinG-family helicase YoaA: its expression and interaction with replication clamp-loader protein HolC in E. coli

2021 ◽  
Author(s):  
Vincent A. Sutera ◽  
Thalia H. Sass ◽  
Scott E. Leonard ◽  
Lingling Wu ◽  
David J. Glass ◽  
...  
Keyword(s):  
Dna Damage ◽  
Mutational Analysis ◽  
Physical Interaction ◽  
Clamp Loader ◽  
Dna Helicases ◽  
E Coli ◽  
C Terminus ◽  
Damage Response ◽  
Domains Of Life

ABSTRACTThe XP-D/DinG family of DNA helicases participate in a variety of ways to preserve genomic stability in all three domains of life. We investigate here the genetic role of one of these proteins,YoaA, of Escherichia coli. In E. coli,YoaA has been identified as having a role in tolerance to the nucleoside azidothymidine (AZT), a DNA replication inhibitor. It is of particular interest because of its physical interaction with a component of the DNA polymerase III holoenzyme, HolC (or χ). We have proposed that this interaction competes with HolC’s interaction with HolD (or ψ) and the rest of the replisome. In this work, we map the residues of YoaA that are required for HolC interaction to the C-terminus of the protein by yeast two-hybrid analysis. We also confirm by gene fusions that YoaA is induced as part of the SOS response to DNA damage and define an upstream “LexA box” sequence in its regulation. Induction of YoaA by AZT is biphasic throughout growth of the culture with an immediate response after treatment and a slower response that peaks in the late log phase of growth. This growth-phase dependent induction by AZT is not blocked by the lexA3 (Ind-) allele, which normally negates its self-cleavage, implying another means to induce the DNA damage response that responds to the nutritional state of the cell.

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 Regulation of Cell Division in Bacteria by Monitoring Genome Integrity and DNA Replication Status

2019 ◽  
Vol 202 (2) ◽  
Author(s):  
Peter E. Burby ◽  
Lyle A. Simmons
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Division ◽  
Dna Replication ◽  
Cell Cycle Progression ◽  
Genome Instability ◽  
Sos Response ◽  
Bacterial Cells ◽  
Cycle Progression ◽  
Content Type

ABSTRACT All organisms regulate cell cycle progression by coordinating cell division with DNA replication status. In eukaryotes, DNA damage or problems with replication fork progression induce the DNA damage response (DDR), causing cyclin-dependent kinases to remain active, preventing further cell cycle progression until replication and repair are complete. In bacteria, cell division is coordinated with chromosome segregation, preventing cell division ring formation over the nucleoid in a process termed nucleoid occlusion. In addition to nucleoid occlusion, bacteria induce the SOS response after replication forks encounter DNA damage or impediments that slow or block their progression. During SOS induction, Escherichia coli expresses a cytoplasmic protein, SulA, that inhibits cell division by directly binding FtsZ. After the SOS response is turned off, SulA is degraded by Lon protease, allowing for cell division to resume. Recently, it has become clear that SulA is restricted to bacteria closely related to E. coli and that most bacteria enforce the DNA damage checkpoint by expressing a small integral membrane protein. Resumption of cell division is then mediated by membrane-bound proteases that cleave the cell division inhibitor. Further, many bacterial cells have mechanisms to inhibit cell division that are regulated independently from the canonical LexA-mediated SOS response. In this review, we discuss several pathways used by bacteria to prevent cell division from occurring when genome instability is detected or before the chromosome has been fully replicated and segregated.

Mutagenesis and More: umuDC and the Escherichia coli SOS Response

Genetics ◽  
1998 ◽  
Vol 148 (4) ◽  
pp. 1599-1610 ◽  
Author(s):  
Bradley T Smith ◽  
Graham C Walker
Keyword(s):  
Escherichia Coli ◽  
Dna Damage ◽  
Cellular Response ◽  
Sos Response ◽  
Posttranslational Regulation ◽  
E Coli ◽  
Sos Mutagenesis ◽  
Induced Mutagenesis ◽  
Mechanistic Basis ◽  
Increase Cell Survival

Abstract The cellular response to DNA damage that has been most extensively studied is the SOS response of Escherichia coli. Analyses of the SOS response have led to new insights into the transcriptional and posttranslational regulation of processes that increase cell survival after DNA damage as well as insights into DNA-damage-induced mutagenesis, i.e., SOS mutagenesis. SOS mutagenesis requires the recA and umuDC gene products and has as its mechanistic basis the alteration of DNA polymerase III such that it becomes capable of replicating DNA containing miscoding and noncoding lesions. Ongoing investigations of the mechanisms underlying SOS mutagenesis, as well as recent observations suggesting that the umuDC operon may have a role in the regulation of the E. coli cell cycle after DNA damage has occurred, are discussed.

scholarly journals 702. Zinc Blockade of SOS Response Inhibits Horizontal Transfer of Antibiotic Resistance Genes in Enteric Bacteria

2018 ◽  
Vol 5 (suppl_1) ◽  
pp. S253-S253
Author(s):  
John Crane ◽  
Mark Sutton ◽  
Muhammad Cheema ◽  
Michael Olyer
Keyword(s):  
Dna Damage ◽  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Horizontal Transfer ◽  
Antibiotic Resistance Genes ◽  
Sos Response ◽  
Enteric Bacteria ◽  
Extracellular Dna ◽  
In Vitro Assays ◽  
E Coli

Abstract Background The SOS response is a conserved response to DNA damage that is found in Gram negative and Gram-positive bacteria. When DNA damage is sustained and severe, activation of error-prone DNA polymerases can induce a higher mutation rate then normally observed, which is called the mutator phenotype or hypermutation. We previously showed that zinc blocked the hypermutation response induced by quinolone antibiotics and mitomycin C in E. coli and Klebsiella pneumoniae (Bunnell BE, Escobar JF, Bair KL, Sutton MD, Crane JK (2017). Zinc blocks SOS-induced antibiotic resistance via inhibition of RecA in Escherichia coli. PLoS ONE 12(5): e0178303. https://doi.org/10.1371/journal.pone.0178303.) In addition to causing copying errors in DNA replication, Beaber et al. showed that induction of the SOS response increased the frequency of horizontal gene transfer into Vibrio cholerae, an organism naturally competent at uptake of extracellular DNA. (Beaber JW, Hochhut B, Waldor MK. 2003. SOS response promotes horizontal dissemination of antibiotic resistance genes. Nature 427:72–74.) Methods. In this study, we tested whether induction of the SOS response could induce transfer of antibiotic resistance from Enterobacter cloacae into E. coli, and whether zinc could inhibit that inter-species transfer of antibiotic resistance. Results. Ciprofloxacin, an inducer of the SOS response, increased the rate of transfer of an extended spectrum β-lactamase (ESBL) gene from Enterobacter into a susceptible E. coli strain. Zinc blocked SOS-induced horizontal transfer of §-lactamase into E. coli. Other divalent metals, such as iron and manganese, failed to inhibit these responses. Conclusion. In vitro assays showed that zinc blocked the ability of RecA to bind to ssDNA, an early step in the SOS response, suggesting the mechanism by which zinc blocks the SOS response. Disclosures All authors: No reported disclosures.

scholarly journals The Main Role of Srs2 in DNA Repair Depends on Its Helicase Activity, Rather than on Its Interactions with PCNA or Rad51

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Alex Bronstein ◽  
Lihi Gershon ◽  
Gilad Grinberg ◽  
Elisa Alonso-Perez ◽  
Martin Kupiec
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Genome Stability ◽  
Dna Helicase ◽  
Main Role ◽  
Helicase Domain ◽  
Helicase Activity ◽  
C Terminus ◽  
Srs2 Helicase

ABSTRACTHomologous recombination (HR) is a mechanism that repairs a variety of DNA lesions. Under certain circumstances, however, HR can generate intermediates that can interfere with other cellular processes such as DNA transcription or replication. Cells have therefore developed pathways that abolish undesirable HR intermediates. TheSaccharomyces cerevisiaeyeast Srs2 helicase has a major role in one of these pathways. Srs2 also works during DNA replication and interacts with the clamp PCNA. The relative importance of Srs2’s helicase activity, Rad51 removal function, and PCNA interaction in genome stability remains unclear. We created a newSRS2allele [srs2(1-850)] that lacks the whole C terminus, containing the interaction site for Rad51 and PCNA and interactions with many other proteins. Thus, the new allele encodes an Srs2 protein bearing only the activity of the DNA helicase. We find that the interactions of Srs2 with Rad51 and PCNA are dispensable for the main role of Srs2 in the repair of DNA damage in vegetative cells and for proper completion of meiosis. On the other hand, it has been shown that in cells impaired for the DNA damage tolerance (DDT) pathways, Srs2 generates toxic intermediates that lead to DNA damage sensitivity; we show that this negative Srs2 activity requires the C terminus of Srs2. Dissection of the genetic interactions of thesrs2(1-850) allele suggest a role for Srs2’s helicase activity in sister chromatid cohesion. Our results also indicate that Srs2’s function becomes more central in diploid cells.IMPORTANCEHomologous recombination (HR) is a key mechanism that repairs damaged DNA. However, this process has to be tightly regulated; failure to regulate it can lead to genome instability. The Srs2 helicase is considered a regulator of HR; it was shown to be able to evict the recombinase Rad51 from DNA. Cells lacking Srs2 exhibit sensitivity to DNA-damaging agents, and in some cases, they display defects in DNA replication. The relative roles of the helicase and Rad51 removal activities of Srs2 in genome stability remain unclear. To address this question, we created a new Srs2 mutant which has only the DNA helicase domain. Our study shows that only the DNA helicase domain is needed to deal with DNA damage and assist in DNA replication during vegetative growth and in meiosis. Thus, our findings shift the view on the role of Srs2 in the maintenance of genome integrity.

scholarly journals Role of HP1β during spermatogenesis and DNA replication

2019 ◽  
Author(s):  
Vijaya Charaka ◽  
Anjana Tiwari ◽  
Raj K Pandita ◽  
Clayton R Hunt ◽  
Tej K. Pandita
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Germ Cells ◽  
Chromatin Condensation ◽  
Genomic Stability ◽  
Replication Forks ◽  
Chromatin Protein ◽  
Male Germ Cells ◽  
Chromatin Compaction

AbstractMaintaining genomic stability in a continually dividing cell population requires accurate DNA repair, especially in male germ cells. Repair and replication protein access to DNA, however, is complicated by chromatin compaction. The HP1β chromatin protein, encoded by Cbx1, is associated with chromatin condensation but its role in meiosis is not clear. To investigate the role of Cbx1 in male germ cells, we generated testis specific Cbx1 deficient transgenic mice by crossing Cbx1flox/flox (Cbx1f/f) mice with Stra8 Cre+/− mice. Loss of Cbx1 in testes adversely affected sperm maturation and Cbx1 deletion increased seminiferous tubule degeneration and basal level DNA damage., We observed that Cbx1−/− MEF cells displayed reduced resolution of stalled DNA replication forks as well as decreased fork restart, indicating defective DNA synthesis. Taken together, these results suggest that loss of Cbx1 in growing cells leads to DNA replication defects and associated DNA damage that impact cell survival.

scholarly journals Deuterium oxide enhances the SOS response of Escherichia coli induced by bactericidal agents

2020 ◽  
pp. 79-80
Author(s):  
С.В. Смирнова ◽  
Т.Н. Шапиро ◽  
Е.В. Игонина ◽  
С.К. Абилев
Keyword(s):  
Escherichia Coli ◽  
Dna Damage ◽  
Nalidixic Acid ◽  
Deuterium Oxide ◽  
Gene Promoter ◽  
Sos Response ◽  
Genotoxic Effect ◽  
E Coli ◽  
Lux Biosensor ◽  
First Time

Изучали генотоксическое действие бактерицидных средств диоксидина, фурацилина и налидиксовой кислоты на клетки дейтерированной культуры lux-биосенсора E.coli MG1655 (pColD::lux), люминесцирующего в результате активации промотора гена колицина colD в ответ на повреждение ДНК. Впервые показано, что оксид дейтерия (D2O) в концентрации 9% усиливает SOS-ответ, индуцированный исследуемыми лекарственными препаратами, в 1,6-2,8 раза в клетках E. coli. We studied the genotoxic effect of bactericidal agents: dioxine, furaciline and nalidixic acid on cells of the deuterated culture lux-biosensor E. coli MG1655 (pColD::lux), which luminesces as a result of activation of the colicin gene promoter colD in response to DNA damage. For the first time, it was shown that deuterium oxide (D2O) at a concentration of 9% increases the SOS response by 1.6-2.8 times in E. coli cells induced by the studied drugs.

scholarly journals SOS and UVM Pathways Have Lesion-Specific Additive and Competing Effects on Mutation Fixation at Replication-Blocking DNA Lesions

1999 ◽  
Vol 181 (5) ◽  
pp. 1515-1523 ◽  
Author(s):  
M. Sayeedur Rahman ◽  
M. Zafri Humayun
Keyword(s):  
Dna Damage ◽  
Transfection Efficiency ◽  
Sos Response ◽  
Genetic Network ◽  
Dna Lesions ◽  
Site Specific ◽  
E Coli ◽  
Mutational Specificity ◽  
Class 1 ◽  
Ap Sites

ABSTRACT Escherichia coli cells have multiple mutagenic pathways that are induced in response to environmental and physiological stimuli. Unlike the well-investigated classical SOS response, little is known about newly recognized pathways such as the UVM (UV modulation of mutagenesis) response. In this study, we compared the contributions of the SOS and UVM pathways on mutation fixation at two representative noninstructive DNA lesions: 3, N4-ethenocytosine (ɛC) and abasic (AP) sites. Because both SOS and UVM responses are induced by DNA damage, and defined UVM-defective E. colistrains are not yet available, we first constructed strains in which expression of the SOS mutagenesis proteins UmuD′ and UmuC (and also RecA in some cases) is uncoupled from DNA damage by being placed under the control of a heterologous lac-derived promoter. M13 single-stranded viral DNA bearing site-specific lesions was transfected into cells induced for the SOS or UVM pathway. Survival effects were determined from transfection efficiency, and mutation fixation at the lesion was analyzed by a quantitative multiplex sequence analysis procedure. Our results suggest that induction of the SOS pathway can independently elevate mutagenesis at both lesions, whereas the UVM pathway significantly elevates mutagenesis at ɛC in an SOS-independent fashion and at AP sites in an SOS-dependent fashion. Although mutagenesis at ɛC appears to be elevated by the induction of either the SOS or the UVM pathway, the mutational specificity profiles for ɛC under SOS and UVM pathways are distinct. Interestingly, when both pathways are active, the UVM effect appears to predominate over the SOS effect on mutagenesis at ɛC, but the total mutation frequency is significantly increased over that observed when each pathway is individually induced. These observations suggest that the UVM response affects mutagenesis not only at class 2 noninstructive lesions (ɛC) but also at classical SOS-dependent (class 1) lesions such as AP sites. Our results add new layers of complexity to inducible mutagenic phenomena: DNA damage activates multiple pathways that have lesion-specific additive as well as suppressive effects on mutation fixation, and some of these pathways are not directly regulated by the SOS genetic network.

Ordered Intracellular Reca-Dna Assemblies

2000 ◽  
Vol 6 (S2) ◽  
pp. 860-861
Author(s):  
S. G. Wolf ◽  
S. Levin-Zaidman ◽  
D. Frenkiel-Krispin ◽  
E. Shimoni ◽  
I. Sabanay ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Electron Micrographs ◽  
Reca Protein ◽  
Sos Response ◽  
Structural Features ◽  
Wild Type ◽  
E Coli ◽  
Dna Damaging Agents

The inducible SOS response increases the ability of bacteria to cope with DNA damage through various DNA repair processes in which the RecA protein plays a central role. We find that induction of the SOS system in wild-type E. coli bacteria results in fast and massive intracellular coaggregation of RecA and DNA into lateral assemblies, which comprise substantial portions of both the cellular RecA and the DNA complement. The structural features of the coaggregates and their relation to in-vitro RecA-DNA are consistent with the possibility that the intracellular assemblies represent a functional entity in which RecA-mediated DNA repair and protection activities occur.Bacterial chromatin is demarcated in electron micrographs of metabolically active cells as amorphous ribosome-free spaces that are irregularly spread over the cytoplasm (Fig. A). Wild-type E. coli cells exposed to DNA-damaging agents that induce the SOS response reveal a strikingly different morphology (Fig. B).

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