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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A new anti conformation for N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (AAF-dG) allows Watson-Crick pairing in the Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4)

Nucleic Acids Research ◽

10.1093/nar/gkj479 ◽

2006 ◽

Vol 34 (3) ◽

pp. 785-795 ◽

Cited By ~ 24

Author(s):

L. Wang

Keyword(s):

Dna Polymerase ◽

Sulfolobus Solfataricus ◽

Anti Conformation ◽

Dna Polymerase Iv

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

10.1101/533422 ◽

2019 ◽

Cited By ~ 2

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.

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Mutagenic nucleotide incorporation and hindered translocation by a food carcinogen C8-dG adduct in Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4): modeling and dynamics studies

Nucleic Acids Research ◽

10.1093/nar/gkl425 ◽

2006 ◽

Vol 34 (11) ◽

pp. 3326-3337 ◽

Cited By ~ 16

Author(s):

L. Zhang

Keyword(s):

Dna Polymerase ◽

Sulfolobus Solfataricus ◽

Nucleotide Incorporation ◽

Food Carcinogen ◽

Dna Polymerase Iv

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

Journal of Bacteriology ◽

10.1128/jb.01481-06 ◽

2006 ◽

Vol 188 (24) ◽

pp. 8573-8585 ◽

Cited By ~ 52

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.

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Replication Bypass of the trans-4-Hydroxynonenal-Derived (6S,8R,11S)-1,N2-Deoxyguanosine DNA Adduct by the Sulfolobus solfataricus DNA Polymerase IV

Chemical Research in Toxicology ◽

10.1021/tx200460j ◽

2012 ◽

Vol 25 (2) ◽

pp. 422-435 ◽

Cited By ~ 7

Author(s):

Surajit Banerjee ◽

Plamen P. Christov ◽

Albena Kozekova ◽

Carmelo J. Rizzo ◽

Martin Egli ◽

...

Keyword(s):

Dna Polymerase ◽

Sulfolobus Solfataricus ◽

Dna Adduct ◽

Dna Polymerase Iv

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Response of Sulfolobus solfataricus Dpo4 polymerase in vitro to a DNA G-quadruplex

Mutagenesis ◽

10.1093/mutage/gez010 ◽

2019 ◽

Vol 34 (3) ◽

pp. 289-297 ◽

Cited By ~ 1

Author(s):

Alexandra Berroyer ◽

Gloria Alvarado ◽

Erik D Larson

Keyword(s):

Repetitive Dna ◽

Dna Sequences ◽

Sulfolobus Solfataricus ◽

Repetitive Dna Sequences ◽

Polymerase Eta ◽

G4 Dna ◽

G Quadruplex ◽

Dna Polymerase Iv ◽

Y Family

Abstract Repetitive DNA sequences support the formation of structures that can interrupt replication and repair, leading to breaks and mutagenesis. One particularly stable structure is G-quadruplex (G4) DNA, which is four-stranded and formed from tandemly repetitive guanine bases. When folded within a template, G4 interferes with DNA synthesis. Similar to non-duplex structures, DNA base lesions can also halt an advancing replication fork, but the Y-family polymerases solve this problem by bypassing the damage. In order to better understand how guanine-rich DNA is replicated, we have investigated the activity of the model Y-family polymerase, Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4), on guanine-rich templates in vitro. We find that Dpo4 progression on templates containing either a single GC-rich hairpin or a G4 DNA structure is greatly reduced and synthesis stalls at the structure. Human polymerase eta (hPol eta) showed the same pattern of stalling at G4; however, and in contrast to Klenow, hPol eta and Dpo4 partially synthesise into the guanine repeat. Substitution of the nucleotide selectivity residue in Dpo4 with alanine permitted ribonucleotide incorporation on unstructured templates, but this further reduced the ability of Dpo4 to synthesise across from the guanine repeats. The advancement of Dpo4 on G4 templates was highest when the reaction was supplied with only deoxycytidine triphosphate, suggesting that high-fidelity synthesis is favoured over misincorporation. Our results are consistent with a model where the Y-family polymerases pause upon encountering G4 structures but have an ability to negotiate some synthesis through tetrad-associated guanines. This suggests that the Y-family polymerases reduce mutagenesis by catalysing the accurate replication of repetitive DNA sequences, but most likely in concert with additional replication and structure resolution activities.

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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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