scholarly journals Modulation of DNA polymerase IV activity by UmuD and RecA* observed by single-molecule time-lapse microscopy

2019 ◽  
Author(s):  
Sarah S. Henrikus ◽  
Amy E. McGrath ◽  
Slobodan Jergic ◽  
Matthew L. Ritger ◽  
Phuong T. Pham ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Polymerase ◽  
Single Molecule ◽  
Imaging Study ◽  
Time Lapse ◽  
Physical Interaction ◽  
Exogenous Dna ◽  
Pol Iv ◽  
Dna Polymerase Iv

AbstractDNA polymerase IV (pol IV) is expressed at increased levels inEscherichia colicells suffering high levels of DNA damage. In a recent single-molecule imaging study, we demonstrated that elevating the pol IV concentration is not sufficient to provide access to binding sites on the nucleoid, suggesting that other factors may recruit pol IV to its substrates once the DNA becomes damaged. Here we extend this work, investigating the proteins UmuD and RecA as potential modulators of pol IV activity. UmuD promotes long-lived association of pol IV with the nucleoid, whereas its cleaved form, UmuD’, which accumulates in DNA-damaged cells, inhibits binding. In agreement with proposed roles for pol IV in homologous recombination, up to 40% of pol IV foci colocalise with a probe for RecA* nucleoprotein filaments in ciprofloxacin-treated cells. A hyperactive RecA mutant,recA(E38K), allows pol IV to bind the nucleoid even in the absence of exogenous DNA damage.In vitro,RecA(E38K) forms RecA*-like structures that can recruit pol IV, even on double-stranded DNA, consistent with a physical interaction between RecA and pol IV. Together, the results indicate that UmuD and RecA modulate the binding of pol IV to its DNA substrates, which frequently coincide with RecA* structures.

scholarly journals Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage

2015 ◽  
Vol 197 (17) ◽  
pp. 2792-2809 ◽  
Author(s):  
Sarita Mallik ◽  
Ellen M. Popodi ◽  
Andrew J. Hanson ◽  
Patricia L. Foster
Keyword(s):  
Dna Damage ◽  
Dna Polymerase ◽  
Dna Helicase ◽  
Dna Lesions ◽  
Replication Forks ◽  
Content Type ◽  
Pol Iv ◽  
Dna Polymerase Iv ◽  
Stalled Replication Forks

ABSTRACTEscherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure ofE. colito DNA-damaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ∼60% of the foci consisted of three Pol IV molecules, while ∼40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that thein vitrointeraction between Rep and Pol IV reported previously also occursin vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ∼70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecAin vivoand is recruited to sites of DSBs to aid in the restoration of DNA replication.IMPORTANCEDNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstratein vivolocalization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings providein vivoevidence that Pol IV aids in maintaining genomic stability not only by bypassing DNA lesions but also by participating in the restoration of stalled replication forks.

scholarly journals Single-molecule live-cell imaging reveals RecB-dependent function of DNA polymerase IV in double strand break repair

2020 ◽  
Vol 48 (15) ◽  
pp. 8490-8508 ◽  
Author(s):  
Sarah S Henrikus ◽  
Camille Henry ◽  
Amy E McGrath ◽  
Slobodan Jergic ◽  
John P McDonald ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Single Molecule ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
E Coli ◽  
Pol Iv ◽  
Dna Polymerase Iv

Abstract Several functions have been proposed for the Escherichia coli DNA polymerase IV (pol IV). Although much research has focused on a potential role for pol IV in assisting pol III replisomes in the bypass of lesions, pol IV is rarely found at the replication fork in vivo. Pol IV is expressed at increased levels in E. coli cells exposed to exogenous DNA damaging agents, including many commonly used antibiotics. Here we present live-cell single-molecule microscopy measurements indicating that double-strand breaks induced by antibiotics strongly stimulate pol IV activity. Exposure to the antibiotics ciprofloxacin and trimethoprim leads to the formation of double strand breaks in E. coli cells. RecA and pol IV foci increase after treatment and exhibit strong colocalization. The induction of the SOS response, the appearance of RecA foci, the appearance of pol IV foci and RecA-pol IV colocalization are all dependent on RecB function. The positioning of pol IV foci likely reflects a physical interaction with the RecA* nucleoprotein filaments that has been detected previously in vitro. Our observations provide an in vivo substantiation of a direct role for pol IV in double strand break repair in cells treated with double strand break-inducing antibiotics.

scholarly journals DNA double-strand breaks induced by reactive oxygen species promote DNA polymerase IV activity inEscherichia coli

2019 ◽  
Author(s):  
Sarah S. Henrikus ◽  
Camille Henry ◽  
John P. McDonald ◽  
Yvonne Hellmich ◽  
Elizabeth A. Wood ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Dna Damage ◽  
Dna Polymerase ◽  
Reactive Oxygen ◽  
Double Strand Breaks ◽  
Oxygen Species ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Pol Iv ◽  
Dna Polymerase Iv

Under many conditions the killing of bacterial cells by antibiotics is potentiated by DNA damage induced by reactive oxygen species (ROS)1–3. A primary cause of ROS-induced cell death is the accumulation of DNA double-strand breaks (DSBs)1,4–6. DNA polymerase IV (pol IV), an error-prone DNA polymerase produced at elevated levels in cells experiencing DNA damage, has been implicated both in ROS-dependent killing and in DSBR7–15. Here, we show using single-molecule fluorescence microscopy that ROS-induced DSBs promote pol IV activity in two ways. First, exposure to the antibiotics ciprofloxacin and trimethoprim triggers an SOS-mediated increase in intracellular pol IV concentrations that is strongly dependent on both ROS and DSBR. Second, in cells that constitutively express pol IV, treatment with an ROS scavenger dramatically reduces the number of pol IV foci formed upon exposure to antibiotics, indicating a role for pol IV in the repair of ROS-induced DSBs.

scholarly journals hSSB2 (NABP1) is required for the recruitment of RPA during the cellular response to DNA UV damage

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Didier Boucher ◽  
Ruvini Kariawasam ◽  
Joshua Burgess ◽  
Adrian Gimenez ◽  
Tristan E. Ocampo ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cellular Response ◽  
Excision Repair ◽  
Protein A ◽  
Ultraviolet B ◽  
Repair System ◽  
Uvb Radiation ◽  
Exogenous Dna

AbstractMaintenance of genomic stability is critical to prevent diseases such as cancer. As such, eukaryotic cells have multiple pathways to efficiently detect, signal and repair DNA damage. One common form of exogenous DNA damage comes from ultraviolet B (UVB) radiation. UVB generates cyclobutane pyrimidine dimers (CPD) that must be rapidly detected and repaired to maintain the genetic code. The nucleotide excision repair (NER) pathway is the main repair system for this type of DNA damage. Here, we determined the role of the human Single-Stranded DNA Binding protein 2, hSSB2, in the response to UVB exposure. We demonstrate that hSSB2 levels increase in vitro and in vivo after UVB irradiation and that hSSB2 rapidly binds to chromatin. Depletion of hSSB2 results in significantly decreased Replication Protein A (RPA32) phosphorylation and impaired RPA32 localisation to the site of UV-induced DNA damage. Delayed recruitment of NER protein Xeroderma Pigmentosum group C (XPC) was also observed, leading to increased cellular sensitivity to UVB. Finally, hSSB2 was shown to have affinity for single-strand DNA containing a single CPD and for duplex DNA with a two-base mismatch mimicking a CPD moiety. Altogether our data demonstrate that hSSB2 is involved in the cellular response to UV exposure.

scholarly journals Comparison of the in Vitro Replication of the 7-(2-Oxoheptyl)-1,N2-etheno-2′-deoxyguanosine and 1,N2-Etheno-2′-deoxyguanosine Lesions bySulfolobus solfataricusP2 DNA Polymerase IV (Dpo4)

2010 ◽  
Vol 23 (8) ◽  
pp. 1330-1341 ◽  
Author(s):  
Plamen P. Christov ◽  
Katya V. Petrova ◽  
Ganesh Shanmugam ◽  
Ivan D. Kozekov ◽  
Albena Kozekova ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
In Vitro Replication ◽  
Dna Polymerase Iv

scholarly journals Role of Pseudomonas aeruginosa dinB-Encoded DNA Polymerase IV in Mutagenesis

2006 ◽  
Vol 188 (24) ◽  
pp. 8573-8585 ◽  
Author(s):  
Laurie H. Sanders ◽  
Andrea Rockel ◽  
Haiping Lu ◽  
Daniel J. Wozniak ◽  
Mark D. Sutton
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Dna Polymerase ◽  
Opportunistic Pathogen ◽  
Binding Assays ◽  
Mutator Phenotype ◽  
Cell Extracts ◽  
Dna Polymerase Iv ◽  
Mechanisms Of Mutagenesis ◽  
Y Family

ABSTRACT Pseudomonas aeruginosa is a human opportunistic pathogen that chronically infects the lungs of cystic fibrosis patients and is the leading cause of morbidity and mortality of people afflicted with this disease. A striking correlation between mutagenesis and the persistence of P. aeruginosa has been reported. In other well-studied organisms, error-prone replication by Y family DNA polymerases contributes significantly to mutagenesis. Based on an analysis of the PAO1 genome sequence, P. aeruginosa contains a single Y family DNA polymerase encoded by the dinB gene. As part of an effort to understand the mechanisms of mutagenesis in P. aeruginosa, we have cloned the dinB gene of P. aeruginosa and utilized a combination of genetic and biochemical approaches to characterize the activity and regulation of the P. aeruginosa DinB protein (DinB Pa ). Our results indicate that DinB Pa is a distributive DNA polymerase that lacks intrinsic proofreading activity in vitro. Modest overexpression of DinB Pa from a plasmid conferred a mutator phenotype in both Escherichia coli and P. aeruginosa. An examination of this mutator phenotype indicated that DinB Pa has a propensity to promote C→A transversions and −1 frameshift mutations within poly(dGMP) and poly(dAMP) runs. The characterization of lexA + and ΔlexA::aacC1 P. aeruginosa strains, together with in vitro DNA binding assays utilizing cell extracts or purified P. aeruginosa LexA protein (LexA Pa ), indicated that the transcription of the dinB gene is regulated as part of an SOS-like response. The deletion of the dinB Pa gene sensitized P. aeruginosa to nitrofurazone and 4-nitroquinoline-1-oxide, consistent with a role for DinB Pa in translesion DNA synthesis over N 2 -dG adducts. Finally, P. aeruginosa exhibited a UV-inducible mutator phenotype that was independent of dinB Pa function and instead required polA and polC, which encode DNA polymerase I and the second DNA polymerase III enzyme, respectively. Possible roles of the P. aeruginosa dinB, polA, and polC gene products in mutagenesis are discussed.

Anti-clastogenic effect of b-glucan extracted from barley towards chemically induced DNA damage in rodent cells

2006 ◽  
Vol 25 (6) ◽  
pp. 319-324 ◽  
Author(s):  
J Pf Angeli ◽  
L R Ribeiro ◽  
M F Bellini ◽  
M S Mantovani
Keyword(s):  
Dna Damage ◽  
Cell Line ◽  
Dna Polymerase ◽  
Mechanism Of Action ◽  
Methylmethane Sulfonate ◽  
Chemically Induced ◽  
Clastogenic Effect ◽  
Chromosomal Aberration Assay ◽  
Mutagenic Mechanism

b-Glucan (BG) was tested in vitro to determine its potential clastogenic and/or anti-clastogenic activity, and attempts were made to elucidate its possible mechanism of action by using combinations with an inhibitor of DNA polymerase. The study was carried out on cells deficient (CHO-k1) and cells proficient (HTC) in phases I and II enzymes, and the DNA damage was assessed by the chromosomal aberration assay. BG did not show a clastogenic effect, but was anti-clastogenic in both cell lines used, and at all concentrations tested (2.5, 5 and 10 mg/mL) in combination with damage inducing agents (methylmethane sulfonate in cell line CHO-k1, and methylmethane sulfonate or 2-aminoanthracene in cell line HTC). BG also showed a protective effect in the presence of a DNA polymerase b inhibitor (cytosine arabinoside-3-phosphate, Ara-C), demonstrating that BG does not act through an anti-mutagenic mechanism of action involving DNA polymerase b.

scholarly journals DNA polymerase X from Deinococcus radiodurans implicated in bacterial tolerance to DNA damage is characterized as a short patch base excision repair polymerase

Microbiology ◽  
2009 ◽  
Vol 155 (9) ◽  
pp. 3005-3014 ◽  
Author(s):  
Nivedita P. Khairnar ◽  
Hari S. Misra
Keyword(s):  
Dna Damage ◽  
Dna Polymerase ◽  
Base Excision Repair ◽  
Deinococcus Radiodurans ◽  
Excision Repair ◽  
Strand Break ◽  
Klenow Fragment ◽  
E Coli ◽  
Base Excision

The Deinococcus radiodurans R1 genome encodes an X-family DNA repair polymerase homologous to eukaryotic DNA polymerase β. The recombinant deinococcal polymerase X (PolX) purified from transgenic Escherichia coli showed deoxynucleotidyltransferase activity. Unlike the Klenow fragment of E. coli, this enzyme showed short patch DNA synthesis activity on heteropolymeric DNA substrate. The recombinant enzyme showed 5′-deoxyribose phosphate (5′-dRP) lyase activity and base excision repair function in vitro, with the help of externally supplied glycosylase and AP endonuclease functions. A polX disruption mutant of D. radiodurans expressing 5′-dRP lyase and a truncated polymerase domain was comparatively less sensitive to γ-radiation than a polX deletion mutant. Both mutants showed higher sensitivity to hydrogen peroxide. Excision repair mutants of E. coli expressing this polymerase showed functional complementation of UV sensitivity. These results suggest the involvement of deinococcal polymerase X in DNA-damage tolerance of D. radiodurans, possibly by contributing to DNA double-strand break repair and base excision repair.

scholarly journals Functional and Physical Interaction of Yeast Mgs1 with PCNA: Impact on RAD6-Dependent DNA Damage Tolerance

2006 ◽  
Vol 26 (14) ◽  
pp. 5509-5517 ◽  
Author(s):  
Takashi Hishida ◽  
Tomoko Ohya ◽  
Yoshino Kubota ◽  
Yusuke Kamada ◽  
Hideo Shinagawa
Keyword(s):  
Dna Damage ◽  
Posttranslational Modifications ◽  
Cell Cycle Progression ◽  
Damage Tolerance ◽  
Genome Instability ◽  
Dna Helicase ◽  
Nuclear Antigen ◽  
Physical Interaction ◽  
Dna Damage Tolerance ◽  
Exogenous Dna

ABSTRACT Proliferating cell nuclear antigen (PCNA), a sliding clamp required for processive DNA synthesis, provides attachment sites for various other proteins that function in DNA replication, DNA repair, cell cycle progression and chromatin assembly. It has been shown that differential posttranslational modifications of PCNA by ubiquitin or SUMO play a pivotal role in controlling the choice of pathway for rescuing stalled replication forks. Here, we explored the roles of Mgs1 and PCNA in replication fork rescue. We provide evidence that Mgs1 physically associates with PCNA and that Mgs1 helps suppress the RAD6 DNA damage tolerance pathway in the absence of exogenous DNA damage. We also show that PCNA sumoylation inhibits the growth of mgs1 rad18 double mutants, in which PCNA sumoylation and the Srs2 DNA helicase coordinately prevent RAD52-dependent homologous recombination. The proposed roles for Mgs1, Srs2, and modified PCNA during replication arrest highlight the importance of modulating the RAD6 and RAD52 pathways to avoid genome instability.

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