How Y-Family DNA polymerase IV is more accurate than Dpo4 at dCTP insertion opposite an N2-dG adduct of benzo[a]pyrene

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.

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Nucleotide selection by the Y-family DNA polymerase Dpo4 involves template translocation and misalignment

Nucleic Acids Research ◽

10.1093/nar/gkt1149 ◽

2013 ◽

Vol 42 (4) ◽

pp. 2555-2563 ◽

Cited By ~ 24

Author(s):

Alfonso Brenlla ◽

Radoslaw P. Markiewicz ◽

David Rueda ◽

Louis J. Romano

Keyword(s):

Dna Polymerase ◽

Single Molecule ◽

Base Pair ◽

Conformational Dynamics ◽

Binary Complex ◽

Translesion Dna Synthesis ◽

Dna Polymerase Iv ◽

Y Family ◽

Dntp Binding ◽

Base Pair Insertion

Abstract Y-family DNA polymerases play a crucial role in translesion DNA synthesis. Here, we have characterized the binding kinetics and conformational dynamics of the Y-family polymerase Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) using single-molecule fluorescence. We find that in the absence of dNTPs, the binary complex shuttles between two different conformations within ∼1 s. These data are consistent with prior crystal structures in which the nucleotide binding site is either occupied by the terminal base pair (preinsertion conformation) or empty following Dpo4 translocation by 1 base pair (insertion conformation). Most interestingly, on dNTP binding, only the insertion conformation is observed and the correct dNTP stabilizes this complex compared with the binary complex, whereas incorrect dNTPs destabilize it. However, if the n+1 template base is complementary to the incoming dNTP, a structure consistent with a misaligned template conformation is observed, in which the template base at the n position loops out. This structure provides evidence for a Dpo4 mutagenesis pathway involving a transient misalignment mechanism.

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Investigating the trade-off between folding and function in a multidomain Y-family DNA polymerase

eLife ◽

10.7554/elife.60434 ◽

2020 ◽

Vol 9 ◽

Author(s):

Xiakun Chu ◽

Zucai Suo ◽

Jin Wang

Keyword(s):

Dna Binding ◽

Dna Polymerase ◽

Protein Interactions ◽

Conformational Dynamics ◽

Dna Lesions ◽

Trade Off ◽

Dna Polymerase Iv ◽

Functional Dna ◽

And Function ◽

Y Family

The way in which multidomain proteins fold has been a puzzling question for decades. Until now, the mechanisms and functions of domain interactions involved in multidomain protein folding have been obscure. Here, we develop structure-based models to investigate the folding and DNA-binding processes of the multidomain Y-family DNA polymerase IV (DPO4). We uncover shifts in the folding mechanism among ordered domain-wise folding, backtracking folding, and cooperative folding, modulated by interdomain interactions. These lead to ‘U-shaped’ DPO4 folding kinetics. We characterize the effects of interdomain flexibility on the promotion of DPO4–DNA (un)binding, which probably contributes to the ability of DPO4 to bypass DNA lesions, which is a known biological role of Y-family polymerases. We suggest that the native topology of DPO4 leads to a trade-off between fast, stable folding and tight functional DNA binding. Our approach provides an effective way to quantitatively correlate the roles of protein interactions in conformational dynamics at the multidomain level.

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Involvement of Y-Family DNA Polymerases in Mutagenesis Caused by Oxidized Nucleotides in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.00281-06 ◽

2006 ◽

Vol 188 (13) ◽

pp. 4992-4995 ◽

Cited By ~ 40

Author(s):

Masami Yamada ◽

Tatsuo Nunoshiba ◽

Masatomi Shimizu ◽

Petr Gruz ◽

Hiroyuki Kamiya ◽

...

Keyword(s):

Escherichia Coli ◽

Dna Polymerase ◽

Dna Polymerases ◽

Dna Polymerase Iv ◽

Y Family

ABSTRACT Escherichia coli DNA polymerase IV incorporated 2-hydroxy-dATP opposite template guanine or thymine and 8-hydroxy-dGTP exclusively opposite adenine in vitro. Mutator phenotypes in sod/fur strains were substantially diminished by deletion of dinB and/or umuDC. DNA polymerases IV and V may be involved in mutagenesis caused by incorporation of the oxidized deoxynucleoside triphosphates.

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Insights into the mismatch discrimination mechanism of Y-family DNA polymerase Dpo4

Biochemical Journal ◽

10.1042/bcj20210162 ◽

2021 ◽

Author(s):

Hunmin Jung ◽

Seongmin Lee

Keyword(s):

Reactive Oxygen Species ◽

Hydrogen Bonds ◽

Dna Polymerase ◽

Sulfolobus Solfataricus ◽

Oxygen Species ◽

Structural Studies ◽

Anti Conformation ◽

Mismatch Discrimination ◽

Dna Polymerase Iv ◽

Y Family

Nucleobases within DNA are attacked by reactive oxygen species to produce 7,8-dihydro-8-oxoguanine (oxoG) and 7,8-dihydro-8-oxoadenine (oxoA) as major oxidative lesions. The high mutagenicity of oxoG is attributed to the lesion’s ability to adopt syn-oxoG:anti-dA with Watson-Crick-like geometry. Recent studies have revealed that Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) inserts nucleotide opposite oxoA in an error-prone manner and accommodates syn-oxoA:anti-dGTP with Watson-Crick-like geometry, highlighting a promutagenic nature of oxoA. To gain further insights into the bypass of oxoA by Dpo4, we have conducted kinetic and structural studies of Dpo4 extending oxoA:dT and oxoA:dG by incorporating dATP opposite templating dT. The extension past oxoA:dG was ~5-fold less efficient than that past oxoA:dT. Structural studies revealed that Dpo4 accommodated dT:dATP base pair past anti-oxoA:dT with little structural distortion. In the Dpo4-oxoA:dG extension structure, oxoA was in an anti conformation and did not form hydrogen bonds with the primer terminus base. Unexpectedely, the dG opposite oxoA exited the primer terminus site and resided in an extrahelical site, where it engaged in minor groove contacts to the two immediate upstream bases. The extrahelical dG conformation appears to be induced by the stabilization of anti-oxoA conformation via bifurcated hydrogen bonds with Arg332. This unprecedented structure suggests that Dpo4 may use Arg332 to sense 8-oxopurines at the primer terminus site and slow the extension from the mismatch by promoting anti conformation of 8-oxopurines.

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Faculty Opinions recommendation of Analysis of translesion replication across an abasic site by DNA polymerase IV of Escherichia coli.

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

10.3410/f.1016604.201051 ◽

2003 ◽

Author(s):

Errol C Friedberg

Keyword(s):

Escherichia Coli ◽

Dna Polymerase ◽

Abasic Site ◽

Dna Polymerase Iv

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Faculty Opinions recommendation of Escherichia coli Rep DNA helicase and error-prone DNA polymerase IV interact physically and functionally.

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

10.3410/f.10652959.11531057 ◽

2011 ◽

Author(s):

Martin Marinus

Keyword(s):

Escherichia Coli ◽

Dna Polymerase ◽

Dna Helicase ◽

Dna Polymerase Iv

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

Journal of Bacteriology ◽

10.1128/jb.00101-15 ◽

2015 ◽

Vol 197 (17) ◽

pp. 2792-2809 ◽

Cited By ~ 20

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.

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