Kinetic analysis of bypass of abasic site by the catalytic core of yeast DNA polymerase eta

Yang, Juntang; Wang, Rong; Liu, Binyan; Xue, Qizhen; Zhong, Mengyu; Zeng, Hao; Zhang, Huidong

doi:10.1016/j.mrfmmm.2015.07.001

Cited by 16 publications

(19 citation statements)

References 30 publications

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“…Generally, incorporation of a correct dNTP by most DNA polymerases shows a biphasic character (burst phase and linear steady-state phase) [ 23 ]. In the burst phase, dNTP is quickly incorporated opposite the template base during the first binding of polymerase to DNA (the first turnover); in the linear steady-state phase, polymerase is dissociated from DNA, then binds to DNA and incorporates dNTP, all of which are limited by the slow dissociation of polymerase from DNA.…”

Section: Resultsmentioning

confidence: 99%

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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: Resultsmentioning

confidence: 99%

“…The samples were then separated on a 20% polyacrylamide (w/v)/7 M urea gel. Products were visualized and quantified using a phosphorimaging screen and Quantity One TM software (Bio-Rad, Hercules, CA, USA) [ 23 ].…”

Section: Methodsmentioning

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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“…Human Pol η also catalyzes efficient and accurate TLS opposite another oxidative lesion, thymine glycol (TG) 9 and can efficiently bypass AP-sites in vitro , by incorporating dATP and dGTP opposite the lesion 2 , 14 , 15 . Yeast Pol η also incorporates dATP or dGTP opposite AP-site, depending on the sequence context 13 , but inefficiently extends the primer beyond the lesion 16 . The role of Pol η in the AP-site bypass in yeast in vivo remains uncertain 17 , 19 , 20 .…”

Section: Introductionmentioning

confidence: 99%

The active site residues Gln55 and Arg73 play a key role in DNA damage bypass by S. cerevisiae Pol η

Boldinova

Ignatov

Kulbachinskiy

et al. 2018

Sci Rep

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Eukaryotic DNA polymerase eta (Pol η) plays a key role in the efficient and accurate DNA translesion synthesis (TLS) opposite UV-induced thymine dimers. Pol η is also involved in bypass of many other DNA lesions but possesses low fidelity on undamaged DNA templates. To better understand the mechanism of DNA synthesis by Pol η we investigated substitutions of evolutionary conserved active site residues Gln55 and Arg73 in Saccharomyces cerevisiae Pol η. We analyzed the efficiency and fidelity of DNA synthesis by the mutant Pol η variants opposite thymine dimers, abasic site, thymine glycol, 8-oxoguanine and on undamaged DNA. Substitutions Q55A and R73A decreased the catalytic activity and significantly affected DNA damage bypass by Pol η. In particular, the Q55A substitution reduced the efficiency of thymine dimers bypass, R73A had a stronger effect on the TLS-activity opposite abasic site, while both substitutions impaired replication opposite thymine glycol. Importantly, the R73A substitution also increased the fidelity of Pol η. Altogether, these results reveal a key role of residues Gln55 and Arg73 in DNA synthesis opposite various types of DNA lesions and highlight the evolutionary importance of the Pol η TLS function at the cost of DNA replication accuracy.

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“…Polη can replicate 8-oxoguanine lesions efficiently and accurately by inserting a C opposite to the damage site (22). Polη can also bypass other lesions such as (6–4) TT photoproducts (23), O-6-methylguanine (24), abasic sites (25), and DNA double-strand breaks (26). In S. cerevisiae , Polη is recruited to stalled replication forks by its physical interaction with monoubiquitinated PCNA (27).…”

Section: Introductionmentioning

confidence: 99%

Lsm12 mediates Pol·q deubiquitination to help Saccharomyces cerevisiae resist oxidative stress

Yao

Shi²,

Chen

et al. 2018

Preprint

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15In Saccharomyces cerevisiae, the Y-family DNA polymerase η (Polη) regulates genome stability 16 in response to different forms of environmental stress by translesion DNA synthesis. To elucidate 17 the role of Polη in oxidative stress-induced DNA damage, we deleted or overexpressed the 18 corresponding gene RAD30, and used transcriptome analysis to screen the potential genes 19 associated with RAD30 to respond to DNA damage. Under 2 mM H 2 O 2 , deletion of RAD30 20 resulted in a 2.2-fold decrease in survival and a 2.8-fold increase in DNA damage, whereas 21 overexpression of RAD30 increased survival and decreased DNA damage by 1.2-and 1.4-fold, 22 IMPORTANCE 34Polη was shown to be critical for cell growth in the yeast Saccharomyces cerevisiae, and deletion 35 of its corresponding gene RAD30 caused a severe growth defect under exposure to oxidative 36 stress with 2 mM H 2 O 2 . Furthermore, we found that Lsm12 physically interacts with Polη and 37 promotes Polη deubiquitination and recruitment. Overall, these findings indicate Lsm12 as a 38 novel regulator mediating Polη deubiquitination that regulates its recruitment in response to 39 DNA damage induced by oxidative stress. 40

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Kinetic analysis of bypass of abasic site by the catalytic core of yeast DNA polymerase eta

Cited by 16 publications

References 30 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

The active site residues Gln55 and Arg73 play a key role in DNA damage bypass by S. cerevisiae Pol η

Lsm12 mediates Pol·q deubiquitination to help Saccharomyces cerevisiae resist oxidative stress

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