Homology modeling of four Y-family, lesion-bypass DNA polymerases: The case that E. coli Pol IV and human Pol κ are orthologs, and E. coli Pol V and human Pol η are orthologs

Irradiation of organisms with UV light produces genotoxic and mutagenic lesions in DNA. Replication through these lesions (translesion DNA synthesis, TSL) in Escherichia coli requires polymerase V (Pol V) and polymerase III (Pol III) holoenzyme. However, some evidence indicates that in the absence of Pol V, and with Pol III inactivated in its proofreading activity by the mutD5 mutation, efficient TSL takes place. The aim of this work was to estimate the involvement of SOS-inducible DNA polymerases, Pol II, Pol IV and Pol V, in UV mutagenesis and in mutation frequency decline (MFD), a mechanism of repair of UV-induced damage to DNA under conditions of arrested protein synthesis. Using the argE3-->Arg(+) reversion to prototrophy system in E. coli AB1157, we found that the umuDC-encoded Pol V is the only SOS-inducible polymerase required for UV mutagenesis, since in its absence the level of Arg(+) revertants is extremely low and independent of Pol II and/or Pol IV. The low level of UV-induced Arg(+) revertants observed in the AB1157mutD5DumuDC strain indicates that under conditions of disturbed proofreading activity of Pol III and lack of Pol V, UV-induced lesions are bypassed without inducing mutations. The presented results also indicate that Pol V may provide substrates for MFD repair; moreover, we suggest that only those DNA lesions which result from umuDC-directed UV mutagenesis are subject to MFD repair.

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Mirror image stereoisomers of the major benzo[a]pyrene N2-dG adduct are bypassed by different lesion-bypass DNA polymerases in E. coli

DNA Repair ◽

10.1016/j.dnarep.2005.12.009 ◽

2006 ◽

Vol 5 (4) ◽

pp. 515-522 ◽

Cited By ~ 37

Author(s):

Kwang Young Seo ◽

Arumugam Nagalingam ◽

Shadi Miri ◽

Jun Yin ◽

Sushil Chandani ◽

...

Keyword(s):

Dna Polymerases ◽

Mirror Image ◽

Lesion Bypass ◽

E Coli

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Enzymatic Switching Between Archaeal DNA Polymerases Facilitates Abasic Site Bypass

Frontiers in Microbiology ◽

10.3389/fmicb.2021.802670 ◽

2021 ◽

Vol 12 ◽

Author(s):

Xu Feng ◽

Baochang Zhang ◽

Ruyi Xu ◽

Zhe Gao ◽

Xiaotong Liu ◽

...

Keyword(s):

Dna Synthesis ◽

Enzyme Kinetics ◽

Dna Polymerases ◽

Dna Lesions ◽

Abasic Site ◽

Lesion Bypass ◽

Translesion Dna Synthesis ◽

Kinetics Studies ◽

Abasic Sites ◽

Y Family

Abasic sites are among the most abundant DNA lesions encountered by cells. Their replication requires actions of specialized DNA polymerases. Herein, two archaeal specialized DNA polymerases were examined for their capability to perform translesion DNA synthesis (TLS) on the lesion, including Sulfolobuss islandicus Dpo2 of B-family, and Dpo4 of Y-family. We found neither Dpo2 nor Dpo4 is efficient to complete abasic sites bypass alone, but their sequential actions promote lesion bypass. Enzyme kinetics studies further revealed that the Dpo4’s activity is significantly inhibited at +1 to +3 site past the lesion, at which Dpo2 efficiently extends the primer termini. Furthermore, their activities are inhibited upon synthesis of 5–6 nt TLS patches. Once handed over to Dpo1, these substrates basically inactivate its exonuclease, enabling the transition from proofreading to polymerization of the replicase. Collectively, by functioning as an “extender” to catalyze further DNA synthesis past the lesion, Dpo2 bridges the activity gap between Dpo4 and Dpo1 in the archaeal TLS process, thus achieving more efficient lesion bypass.

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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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Structure-based interpretation of missense mutations in Y-family DNA polymerases and their implications for polymerase function and lesion bypass

DNA Repair ◽

10.1016/s1568-7864(02)00019-8 ◽

2002 ◽

Vol 1 (5) ◽

pp. 343-358 ◽

Cited By ~ 53

Author(s):

F Boudsocq

Keyword(s):

Dna Polymerases ◽

Missense Mutations ◽

Lesion Bypass ◽

Y Family

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MsDpo4—a DinB Homolog fromMycobacterium smegmatis—Is an Error-Prone DNA Polymerase That Can Promote G:T and T:G Mismatches

Journal of Nucleic Acids ◽

10.1155/2012/285481 ◽

2012 ◽

Vol 2012 ◽

pp. 1-8 ◽

Cited By ~ 13

Author(s):

Amit Sharma ◽

Deepak T. Nair

Keyword(s):

Mycobacterium Tuberculosis ◽

Dna Polymerase ◽

Environmental Conditions ◽

Mycobacterium Smegmatis ◽

Molecular Level ◽

Dna Polymerases ◽

Adaptive Mutagenesis ◽

E Coli ◽

Nucleotide Incorporation ◽

Y Family

Error-prone DNA synthesis in prokaryotes imparts plasticity to the genome to allow for evolution in unfavorable environmental conditions, and this phenomenon is termed adaptive mutagenesis. At a molecular level, adaptive mutagenesis is mediated by upregulating the expression of specialized error-prone DNA polymerases that generally belong to the Y-family, such as the polypeptide product of thedinBgene in case ofE. coli. However, unlikeE. coli, it has been seen that expression of the homologs ofdinBinMycobacterium tuberculosisare not upregulated under conditions of stress. These studies suggest that DinB homologs inMycobacteriamight not be able to promote mismatches and participate in adaptive mutagenesis. We show that a representative homolog fromMycobacterium smegmatis(MsDpo4) can carry out template-dependent nucleotide incorporation and therefore is a DNA polymerase. In addition, it is seen that MsDpo4 is also capable of misincorporation with a significant ability to promote G:T and T:G mismatches. The frequency of misincorporation for these two mismatches is similar to that exhibited by archaeal and prokaryotic homologs. Overall, our data show that MsDpo4 has the capacity to facilitate transition mutations and can potentially impart plasticity to the genome.

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Quantitative analysis of the efficiency and mutagenic spectra of abasic lesion bypass catalyzed by human Y-family DNA polymerases

Nucleic Acids Research ◽

10.1093/nar/gkq719 ◽

2010 ◽

Vol 39 (2) ◽

pp. 609-622 ◽

Cited By ~ 20

Author(s):

Shanen M. Sherrer ◽

Kevin A. Fiala ◽

Jason D. Fowler ◽

Sean A. Newmister ◽

John M. Pryor ◽

...

Keyword(s):

Quantitative Analysis ◽

Dna Polymerases ◽

Lesion Bypass ◽

Y Family

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Architecture of Y-Family DNA Polymerases Relevant to Translesion DNA Synthesis as Revealed in Structural and Molecular Modeling Studies

Journal of Nucleic Acids ◽

10.4061/2010/784081 ◽

2010 ◽

Vol 2010 ◽

pp. 1-20 ◽

Cited By ~ 8

Author(s):

Sushil Chandani ◽

Christopher Jacobs ◽

Edward L. Loechler

Keyword(s):

Conformational Changes ◽

Dna Polymerases ◽

Class Versus ◽

Lesion Bypass ◽

Translesion Dna Synthesis ◽

Protein Conformational Changes ◽

Modeling Studies ◽

Hydrophobic Amino Acids ◽

Molecular Modeling Studies ◽

Y Family

DNA adducts, which block replicative DNA polymerases (DNAPs), are often bypassed by lesion-bypass DNAPs, which are mostly in the Y-Family. Y-Family DNAPs can do non-mutagenic or mutagenic dNTP insertion, and understanding this difference is important, because mutations transform normal into tumorigenic cells. Y-Family DNAP architecture that dictates mechanism, as revealed in structural and modeling studies, is considered. Steps from adduct blockage of replicative DNAPs, to bypass by a lesion-bypass DNAP, to resumption of synthesis by a replicative DNAP are described. Catalytic steps and protein conformational changes are considered. One adduct is analyzed in greater detail: the major benzo[a]pyrene adduct , which is bypassed non-mutagenically (dCTP insertion) by Y-family DNAPs in the IV/-class and mutagenically (dATP insertion) by V/-class Y-Family DNAPs. Important architectural differences between IV/-class versus V/-class DNAPs are discussed, including insights gained by analyzing ~400 sequences each for bacterial DNAPs IV and V, along with sequences from eukaryotic DNAPs kappa, eta and iota. The little finger domains of Y-Family DNAPs do not show sequence conservation; however, their structures are remarkably similar due to the presence of a core of hydrophobic amino acids, whose exact identity is less important than the hydrophobic amino acid spacing.

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Nucleoside Analogues Are Potent Inducers of Pol V-mediated Mutagenesis

Biomolecules ◽

10.3390/biom11060843 ◽

2021 ◽

Vol 11 (6) ◽

pp. 843

Author(s):

Balagra Kasim Sumabe ◽

Synnøve Brandt Ræder ◽

Lisa Marie Røst ◽

Animesh Sharma ◽

Eric S. Donkor ◽

...

Keyword(s):

Mammalian Cells ◽

Mutation Frequency ◽

Nucleoside Analogues ◽

Cancer Therapies ◽

E Coli ◽

Replicative Stress ◽

Pol V ◽

Dna And Rna ◽

Sos Induction ◽

Anti Cancer

Drugs targeting DNA and RNA in mammalian cells or viruses can also affect bacteria present in the host and thereby induce the bacterial SOS system. This has the potential to increase mutagenesis and the development of antimicrobial resistance (AMR). Here, we have examined nucleoside analogues (NAs) commonly used in anti-viral and anti-cancer therapies for potential effects on mutagenesis in Escherichia coli, using the rifampicin mutagenicity assay. To further explore the mode of action of the NAs, we applied E. coli deletion mutants, a peptide inhibiting Pol V (APIM-peptide) and metabolome and proteome analyses. Five out of the thirteen NAs examined, including three nucleoside reverse transcriptase inhibitors (NRTIs) and two anti-cancer drugs, increased the mutation frequency in E. coli by more than 25-fold at doses that were within reported plasma concentration range (Pl.CR), but that did not affect bacterial growth. We show that the SOS response is induced and that the increase in mutation frequency is mediated by the TLS polymerase Pol V. Quantitative mass spectrometry-based metabolite profiling did not reveal large changes in nucleoside phosphate or other central carbon metabolite pools, which suggests that the SOS induction is an effect of increased replicative stress. Our results suggest that NAs/NRTIs can contribute to the development of AMR and that drugs inhibiting Pol V can reverse this mutagenesis.

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