Kinetic analysis of bypass of O6- methylguanine by the catalytic core of yeast DNA polymerase eta

Liu, Binyan; Xue, Qizheng; Gu, Shiling; Wang, Weiping; Chen, Jie; Li, Ying-Qing; Wang, Chunxue; Zhang, Huidong

doi:10.1016/j.abb.2016.03.009

Cited by 17 publications

(8 citation statements)

References 39 publications

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“…The exonuclease activity of Gp90 should be eliminated to analyze 8-oxoG bypass by Gp90 kinetically. In our previous results, Glu-60, Asp-137, and Asp-234 were predicted as the exonuclease active residues of Gp90 [ 29 ]. Gp90 D234A, in which Asp-234 was replaced with Ala, can efficiently abolish the ssDNA and dsDNA exonuclease activities while maintaining its polymerase activity ( Figure 1 ).…”

Section: Discussionmentioning

confidence: 99%

Error-Free Bypass of 7,8-dihydro-8-oxo-2′-deoxyguanosine by DNA Polymerase of Pseudomonas aeruginosa Phage PaP1

Xue

Liu

et al. 2017

Genes

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As one of the most common forms of oxidative DNA damage, 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxoG) generally leads to G:C to T:A mutagenesis. To study DNA replication encountering 8-oxoG by the sole DNA polymerase (Gp90) of Pseudomonas aeruginosa phage PaP1, we performed steady-state and pre-steady-state kinetic analyses of nucleotide incorporation opposite 8-oxoG by Gp90 D234A that lacks exonuclease activities on ssDNA and dsDNA substrates. Gp90 D234A could bypass 8-oxoG in an error-free manner, preferentially incorporate dCTP opposite 8-oxoG, and yield similar misincorporation frequency to unmodified G. Gp90 D234A could extend beyond C:8-oxoG or A:8-oxoG base pairs with the same efficiency. dCTP incorporation opposite G and dCTP or dATP incorporation opposite 8-oxoG showed fast burst phases. The burst of incorporation efficiency (kpol/Kd,dNTP) is decreased as dCTP:G > dCTP:8-oxoG > dATP:8-oxoG. The presence of 8-oxoG in DNA does not affect its binding to Gp90 D234A in a binary complex but it does affect it in a ternary complex with dNTP and Mg2+, and dATP misincorporation opposite 8-oxoG further weakens the binding of Gp90 D234A to DNA. This study reveals Gp90 D234A can bypass 8-oxoG in an error-free manner, providing further understanding in DNA replication encountering oxidation lesion for P.aeruginosa phage PaP1.

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Section: Discussionmentioning

confidence: 99%

Error-Free Bypass of 7,8-dihydro-8-oxo-2′-deoxyguanosine by DNA Polymerase of Pseudomonas aeruginosa Phage PaP1

Xue

Liu

et al. 2017

Genes

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“…Steady-state experiments of single-base incorporation and one-base extension were performed by adding a single dNTP at various concentrations. The molar ratio of Dpo4 to DNA was 1:10 (2 nM polymerase and 20 nM DNA) (62,63). For steady-state single-base incorporation, wild-type or A181D mutated Dpo4 and DNA were mixed in the buffer mentioned for the fidelity assay.…”

Section: Methodsmentioning

confidence: 99%

Increased Processivity, Misincorporation, and Nucleotide Incorporation Efficiency in Sulfolobus solfataricus Dpo4 Thumb Domain Mutants

Wang

Liang

et al. 2017

Appl Environ Microbiol

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The present study aimed to increase the processivity of DNA polymerase Dpo4. Protein engineering and bioinformatics were used to compile a library of potential Dpo4 mutation sites. Ten potential mutants were identified and constructed. A primer extension assay was used to evaluate the processivity of Dpo4 mutants. Thumb (A181D) and finger (E63K) domain mutants showed a processivity of 20 and 19 nucleotides (nt), respectively. A little finger domain mutant (I248Y) exhibited a processivity of 17 nt, only 1 nt more than wild-type Dpo4. Furthermore, the A181D mutant showed lower fidelity and higher nucleotide incorporation efficiency (4.74 × 10 s μM) than E63K and I248Y mutants. When tasked with bypassing damage, the A181D mutant exhibited a 3.81-fold and 2.62-fold higher catalytic efficiency (/ ) at incorporating dCTP and dATP, respectively, than wild-type Dpo4. It also showed a 55% and 91.5% higher catalytic efficiency when moving beyond the damaged 8-oxoG:C and 8-oxoG:A base pairs, respectively, compared to wild-type Dpo4. Protein engineering and bioinformatics methods can effectively increase the processivity and translesion synthesis ability of Dpo4. DNA polymerases with poor fidelity can be exploited to store data and record changes in response to the intracellular environment. Dpo4 is such an enzyme, although its use is hindered by its low processivity. In this work, we used a bioinformatics and protein engineering approach to generate Dpo4 mutants with improved processivity. We identified the Dpo4 thumb domain as the most relevant in controlling processivity.

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“…The products were visualized by phosphorimaging and quantified by use of Quantity One software. [2,43] The 20-mer,2 7-mer,3 0-mer,3 3-mer,a nd 62-mer products were used to identify the extension bands. The concentrations of all the products from 28-mer to 62-mer (bypass products) were summed.…”

Section: Oligonucleotidementioning

confidence: 99%

Protein Interactions in the T7 DNA Replisome Facilitate DNA Damage Bypass

et al. 2018

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The DNA replisome inevitably encounters DNA damage during DNA replication. The T7 DNA replisome contains a DNA polymerase (gp5), the processivity factor thioredoxin (trx), a helicase-primase (gp4), and a ssDNA-binding protein (gp2.5). T7 protein interactions mediate this DNA replication. However, whether the protein interactions could promote DNA damage bypass is still little addressed. In this study, we investigated strand-displacement DNA synthesis past 8-oxoG or O -MeG lesions at the synthetic DNA fork by the T7 DNA replisome. DNA damage does not obviously affect the binding affinities between helicase, polymerase, and DNA fork. Relative to unmodified G, both 8-oxoG and O -MeG-as well as GC-rich template sequence clusters-inhibit strand-displacement DNA synthesis and produce partial extension products. Relative to the gp4 ΔC-tail, gp4 promotes DNA damage bypass. The presence of gp2.5 also promotes it. Thus, the interactions of polymerase with helicase and ssDNA-binding protein facilitate DNA damage bypass. Accessory proteins in other complicated DNA replisomes also facilitate bypassing DNA damage in similar manner. This work provides new mechanistic information relating to DNA damage bypass by the DNA replisome.

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Kinetic analysis of bypass of O6- methylguanine by the catalytic core of yeast DNA polymerase eta

Cited by 17 publications

References 39 publications

Error-Free Bypass of 7,8-dihydro-8-oxo-2′-deoxyguanosine by DNA Polymerase of Pseudomonas aeruginosa Phage PaP1

Error-Free Bypass of 7,8-dihydro-8-oxo-2′-deoxyguanosine by DNA Polymerase of Pseudomonas aeruginosa Phage PaP1

Increased Processivity, Misincorporation, and Nucleotide Incorporation Efficiency in Sulfolobus solfataricus Dpo4 Thumb Domain Mutants

Protein Interactions in the T7 DNA Replisome Facilitate DNA Damage Bypass

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