Role of the Y-Family DNA Polymerases YqjH and YqjW in Protecting Sporulating Bacillus subtilis Cells from DNA Damage

AbstractDNA replication forks are intrinsically asymmetric and may arrest during the cell cycle upon encountering modifications in the DNA. We have studied real time dynamics of three DNA polymerases and an exonuclease at a single molecule level in the bacterium Bacillus subtilis. PolC and DnaE work in a symmetric manner and show similar dwell times. After addition of DNA damage, their static fractions and dwell times decreased, in agreement with increased re-establishment of replication forks. Only a minor fraction of replication forks showed a loss of active polymerases, indicating relatively robust activity during DNA repair. Conversely, PolA, homolog of polymerase I and exonuclease ExoR were rarely present at forks during unperturbed replication but were recruited to replications forks after induction of DNA damage. Protein dynamics of PolA or ExoR were altered in the absence of each other during exponential growth and during DNA repair, indicating overlapping functions. Purified ExoR displayed exonuclease activity and preferentially bound to DNA having 5′ overhangs in vitro. Our analyses support the idea that two replicative DNA polymerases work together at the lagging strand whilst only PolC acts at the leading strand, and that PolA and ExoR perform inducible functions at replication forks during DNA repair.

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Role of specialized DNA polymerases in the limitation of replicative stress and DNA damage transmission

Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis ◽

10.1016/j.mrfmmm.2017.08.002 ◽

2018 ◽

Vol 808 ◽

pp. 62-73 ◽

Cited By ~ 10

Author(s):

Elodie Bournique ◽

Marina Dall’Osto ◽

Jean-Sébastien Hoffmann ◽

Valérie Bergoglio

Keyword(s):

Dna Damage ◽

Dna Polymerases ◽

Replicative Stress

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Role of the Nfo (YqfS) and ExoA Apurinic/Apyrimidinic Endonucleases in Protecting Bacillus subtilis Spores from DNA Damage

Journal of Bacteriology ◽

10.1128/jb.187.21.7374-7381.2005 ◽

2005 ◽

Vol 187 (21) ◽

pp. 7374-7381 ◽

Cited By ~ 25

Author(s):

José M. Salas-Pacheco ◽

Barbara Setlow ◽

Peter Setlow ◽

Mario Pedraza-Reyes

Keyword(s):

Dna Damage ◽

Bacillus Subtilis ◽

Spontaneous Mutation ◽

Wild Type ◽

Strand Breaks ◽

Dry Heat ◽

Wet Heat ◽

Ap Sites ◽

Dna Strand

ABSTRACT The Bacillus subtilis enzymes ExoA and Nfo (originally termed YqfS) are endonucleases that can repair apurinic/apyrimidinic (AP) sites and strand breaks in DNA. We have analyzed how the lack of ExoA and Nfo affects the resistance of growing cells and dormant spores of B. subtilis to a variety of treatments, some of which generate AP sites and DNA strand breaks. The lack of ExoA and Nfo sensitized spores (termed α−β−) lacking the majority of their DNA-protective α/β-type small, acid-soluble spore proteins (SASP) to wet heat. However, the lack of these enzymes had no effect on the wet-heat resistance of spores that retained α/β-type SASP. The lack of either ExoA or Nfo sensitized wild-type spores to dry heat, but loss of both proteins was necessary to sensitize α−β− spores to dry heat. The lack of ExoA and Nfo also sensitized α−β−, but not wild-type, spores to desiccation. In contrast, loss of ExoA and Nfo did not sensitize growing cells or wild-type or α−β− spores to hydrogen peroxide or t-butylhydroperoxide. Loss of ExoA and Nfo also did not increase the spontaneous mutation frequency of growing cells. exoA expression took place not only in growing cells, but also in the forespore compartment of the sporulating cell. These results, together with those from previous work, suggest that ExoA and Nfo are additional factors that protect B. subtilis spores from DNA damage accumulated during spore dormancy.

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Role of Dipicolinic Acid in Resistance and Stability of Spores of Bacillus subtilis with or without DNA-Protective α/β-Type Small Acid-Soluble Proteins

Journal of Bacteriology ◽

10.1128/jb.00212-06 ◽

2006 ◽

Vol 188 (11) ◽

pp. 3740-3747 ◽

Cited By ~ 114

Author(s):

Barbara Setlow ◽

Swaroopa Atluri ◽

Ryan Kitchel ◽

Kasia Koziol-Dube ◽

Peter Setlow

Keyword(s):

Hydrogen Peroxide ◽

Dna Damage ◽

Bacillus Subtilis ◽

Dipicolinic Acid ◽

Dry Weight ◽

Definitive Evidence ◽

Dry Heat ◽

Spore Resistance ◽

Wet Heat

ABSTRACT Dipicolinic acid (DPA) comprises ∼10% of the dry weight of spores of Bacillus species. Although DPA has long been implicated in spore resistance to wet heat and spore stability, definitive evidence on the role of this abundant molecule in spore properties has generally been lacking. Bacillus subtilis strain FB122 (sleB spoVF) produced very stable spores that lacked DPA, and sporulation of this strain with DPA yielded spores with nearly normal DPA levels. DPA-replete and DPA-less FB122 spores had similar levels of the DNA protective α/β-type small acid-soluble spore proteins (SASP), but the DPA-less spores lacked SASP-γ. The DPA-less FB122 spores exhibited similar UV resistance to the DPA-replete spores but had lower resistance to wet heat, dry heat, hydrogen peroxide, and desiccation. Neither wet heat nor hydrogen peroxide killed the DPA-less spores by DNA damage, but desiccation did. The inability to synthesize both DPA and most α/β-type SASP in strain PS3664 (sspA sspB sleB spoVF) resulted in spores that lost viability during sporulation, at least in part due to DNA damage. DPA-less PS3664 spores were more sensitive to wet heat than either DPA-less FB122 spores or DPA-replete PS3664 spores, and the latter also retained viability during sporulation. These and previous results indicate that, in addition to α/β-type SASP, DPA also is extremely important in spore resistance and stability and, further, that DPA has some specific role(s) in protecting spore DNA from damage. Specific roles for DPA in protecting spore DNA against damage may well have been a major driving force for the spore's accumulation of the high levels of this small molecule.

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Faculty Opinions recommendation of Investigating the role of the little finger domain of Y-family DNA polymerases in low fidelity synthesis and translesion replication.

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

10.3410/f.1019694.223472 ◽

2004 ◽

Author(s):

Errol C Friedberg

Keyword(s):

Dna Polymerases ◽

Y Family ◽

Little Finger

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The Roles of UmuD in Regulating Mutagenesis

Journal of Nucleic Acids ◽

10.4061/2010/947680 ◽

2010 ◽

Vol 2010 ◽

pp. 1-12 ◽

Cited By ~ 8

Author(s):

Jaylene N. Ollivierre ◽

Jing Fang ◽

Penny J. Beuning

Keyword(s):

Dna Damage ◽

Protein Interactions ◽

Dna Polymerases ◽

Gene Products ◽

E Coli ◽

Domains Of Life ◽

Induced Mutagenesis ◽

Multiple Conformations ◽

Y Family ◽

Damaged Dna

All organisms are subject to DNA damage from both endogenous and environmental sources. DNA damage that is not fully repaired can lead to mutations. Mutagenesis is now understood to be an active process, in part facilitated by lower-fidelity DNA polymerases that replicate DNA in an error-prone manner. Y-family DNA polymerases, found throughout all domains of life, are characterized by their lower fidelity on undamaged DNA and their specialized ability to copy damaged DNA. TwoE. coliY-family DNA polymerases are responsible for copying damaged DNA as well as for mutagenesis. These DNA polymerases interact with different forms of UmuD, a dynamic protein that regulates mutagenesis. The UmuD gene products, regulated by the SOS response, exist in two principal forms:UmuD2, which prevents mutagenesis, andUmuD2′, which facilitates UV-induced mutagenesis. This paper focuses on the multiple conformations of the UmuD gene products and how their protein interactions regulate mutagenesis.

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Role of the DinB Homologs Rv1537 and Rv3056 in Mycobacterium tuberculosis

Journal of Bacteriology ◽

10.1128/jb.01135-09 ◽

2010 ◽

Vol 192 (8) ◽

pp. 2220-2227 ◽

Cited By ~ 41

Author(s):

Bavesh D. Kana ◽

Garth L. Abrahams ◽

Nackmoon Sung ◽

Digby F. Warner ◽

Bhavna G. Gordhan ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Alkylating Agents ◽

Spontaneous Mutation ◽

Dna Polymerases ◽

Binding Motif ◽

Dependent Manner ◽

Mouse Macrophages ◽

Y Family

ABSTRACT The environment encountered by Mycobacterium tuberculosis during infection is genotoxic. Most bacteria tolerate DNA damage by engaging specialized DNA polymerases that catalyze translesion synthesis (TLS) across sites of damage. M. tuberculosis possesses two putative members of the DinB class of Y-family DNA polymerases, DinB1 (Rv1537) and DinB2 (Rv3056); however, their role in damage tolerance, mutagenesis, and survival is unknown. Here, both dinB1 and dinB2 are shown to be expressed in vitro in a growth phase-dependent manner, with dinB2 levels 12- to 40-fold higher than those of dinB1. Yeast two-hybrid analyses revealed that DinB1, but not DinB2, interacts with the β-clamp, consistent with its canonical C-terminal β-binding motif. However, knockout of dinB1, dinB2, or both had no effect on the susceptibility of M. tuberculosis to compounds that form N 2-dG adducts and alkylating agents. Similarly, deletion of these genes individually or in combination did not affect the rate of spontaneous mutation to rifampin resistance or the spectrum of resistance-conferring rpoB mutations and had no impact on growth or survival in human or mouse macrophages or in mice. Moreover, neither gene conferred a mutator phenotype when expressed ectopically in Mycobacterium smegmatis. The lack of the effect of altering the complements or expression levels of dinB1 and/or dinB2 under conditions predicted to be phenotypically revealing suggests that the DinB homologs from M. tuberculosis do not behave like their counterparts from other organisms.

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