The Ability of a Variety of Polymerases to Synthesize Past Site-specific cis-syn, trans-syn-II, (6–4), and Dewar Photoproducts of Thymidylyl-(3′→5′)-thymidine

Smith, Colin A.; Baeten, Jared M.; Taylor, John‐Stephen

doi:10.1074/jbc.273.34.21933

Cited by 61 publications

(51 citation statements)

References 95 publications

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“…This holds true for damage-containing templates as shown with the T7 pol and the E. coli pol I, pol II, and pol III enzymes (67)(68)(69)(70). Thus, if DnaE has a proofreader, it is likely that the DnaE-proofreader complex exhibits a lower bypass capacity than described here for the purified DnaE protein.…”

Section: Dual Function Of Polymerase Dnaementioning

confidence: 79%

Involvement of DnaE, the Second Replicative DNA Polymerase from Bacillus subtilis, in DNA Mutagenesis

Bécherel¹,

d’Alençon²,

Canceill³

et al. 2004

Journal of Biological Chemistry

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In a large group of organisms including low G ؉ C bacteria and eukaryotic cells, DNA synthesis at the replication fork strictly requires two distinct replicative DNA polymerases. These are designated pol C and DnaE in Bacillus subtilis. We recently proposed that DnaE might be preferentially involved in lagging strand synthesis, whereas pol C would mainly carry out leading strand synthesis. The biochemical analysis of DnaE reported here is consistent with its postulated function, as it is a highly potent enzyme, replicating as fast as 240 nucleotides/s, and stalling for more than 30 s when encountering annealed 5-DNA end. DnaE is devoid of 3 3 5-proofreading exonuclease activity and has a low processivity (1-75 nucleotides), suggesting that it requires additional factors to fulfill its role in replication. Interestingly, we found that (i) DnaE is SOS-inducible; (ii) variation in DnaE or pol C concentration has no effect on spontaneous mutagenesis; (iii) depletion of pol C or DnaE prevents UV-induced mutagenesis; and (iv) purified DnaE has a rather relaxed active site as it can bypass lesions that generally block other replicative polymerases. These results suggest that DnaE and possibly pol C have a function in DNA repair/mutagenesis, in addition to their role in DNA replication.In all living organisms, DNA replication is carried out by a functionally highly conserved protein complex. Genetic and biochemical data have shown that this complex, called DNA polymerase holoenzyme, contains two copies of an essential replicative DNA polymerase in Escherichia coli, T4 and T7 phages, and SV40 (reviewed in Refs. 1-6). In contrast, replication requires two different polymerases in bacteria Bacillus subtilis and Staphylococcus aureus (pol 1 C and DnaE, C family (7, 8)) and in eukaryotes including Saccharomyces cerevisiae, Xenopus, and human (pol ␦ and pol ⑀, B family; reviewed in Refs. 5 and 9 -11). Thus, holoenzyme of these organisms might be more complex, containing two different polymerases instead of two copies of a single polymerase. This higher level of complexity would hold true for many organisms as follows: (i) systematic sequencing of bacterial genomes (more than 100 completed to date) revealed that ϳ50% carry at least two copies of dnaE or contain dnaE and polC (no genome containing only polC has been detected so far), and (ii) pol ␦ and pol ⑀ seem to be ubiquitous in eukaryotes. It is well established that pol C in bacteria and pol ␦ in eukaryotes are required at the replication fork (5, 9 -15). On the other hand, the specific roles of DnaE and pol ⑀ during replication are still not known. In B. subtilis, it was reported that the purified DnaE protein has a DNA polymerase activity devoid of proofreading activity and presents a high affinity for dNTP (14,16). Genetic and cytological data as well as in vivo assays of radioactive precursor incorporation have shown that DnaE, like pol C, is essential for the elongation phase of replication and is associated with the replication factory at mid-cell (7). Moreover, stu...

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Section: Dual Function Of Polymerase Dnaementioning

confidence: 79%

Involvement of DnaE, the Second Replicative DNA Polymerase from Bacillus subtilis, in DNA Mutagenesis

Bécherel¹,

d’Alençon²,

Canceill³

et al. 2004

Journal of Biological Chemistry

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“…Biochemical and structural studies with the T7 Pol have shown that this Pol inserts an A opposite the 3Ј T of a CPD with the photoproduct lying outside the active site, whereas insertion opposite the 5Ј T occurs with the CPD in the active site (31,32,34). Thus, the A incorporation opposite the 3Ј T of a CPD is not template-directed but rather occurs via an abasic site-like intermediate.…”

Section: Discussionmentioning

confidence: 99%

Distinct mechanisms of cis-syn thymine dimer bypass by Dpo4 and DNA polymerase η

Johnson

Prakash²,

Prakash³

2005

Proc. Natl. Acad. Sci. U.S.A.

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UV-light-induced cyclobutane pyrimidine dimers (CPDs) present a severe block to synthesis by replicative DNA polymerases (Pols), whereas Pol promotes proficient and error-free replication through CPDs. Although the archael Dpo4, which, like Pol , belongs to the Y family of DNA Pols, can also replicate through a CPD, it is much less efficient than Pol . The x-ray crystal structure of Dpo4 complexed with either the 3 -thymine (T) or the 5 T of a cis-syn TT dimer has indicated that, whereas the 3 T of the dimer forms a Watson-Crick base pair with the incoming dideoxy ATP, the 5 T forms a Hoogsteen base pair with the dideoxy ATP in syn conformation. Based upon these observations, a similar mechanism involving Hoogsteen base pairing of the 5 T of the dimer with the incoming A has been proposed for Pol . Here we examine the mechanisms of CPD bypass by Dpo4 and Pol using nucleotide analogs that specifically disrupt the which cyclobutane pyrimidine dimers (CPDs) are the most prevalent form, and which, if not removed, lead to mutations and cancer formation. Although a cis-syn thymine-thymine (TT) dimer introduces only a modest deformation of the DNA helix, bending it by Ϸ30°and unwinding it by Ϸ9°, and the ability of the two Ts in the dimer to form Watson-Crick base pairs with the As is not significantly altered (1-4), a CPD still presents a strong block to synthesis by most DNA polymerases (Pols), because the covalent linkage of the two Ts in the CPD prevents the kinking of the DNA backbone so that the 5Ј T of the dimer cannot be pushed out of the active site. This then prevents the classical replicative and repair Pols from replicating through this lesion, because they can accommodate only a single templating nucleotide in their active site (5-7), which provides for a tight fit of the template base with the correct incoming nucleotide and thereby imposes a high degree of geometric selectivity.Because of the steric constraints imposed by the active site, the binding pocket of high-fidelity replicative and repair DNA Pols accommodates only the base pairs with correct Watson-Crick geometry (8). Consequently, these Pols are inhibited from replicating through DNA lesions that distort the Watson-Crick base-pairing geometry. The Y family DNA Pols, on the other hand, can synthesize past DNA lesions, but they synthesize DNA with much lower fidelity and processivity than the replicative͞ repair Pols. The eukaryotic Y family Pols, however, exhibit a high degree of specificity in their lesion bypass abilities (9), and of these, Pol has the unique ability to replicate through CPDs proficiently and accurately (10, 11). Consequently, inactivation of Pol in both yeast and humans results in increased UV light mutagenesis (12-15), and defects in Pol in humans cause the skin cancer-prone syndrome, the variant form of xeroderma pigmentosum (11, 16). Hence, Pol prevents skin cancers by reducing the incidence of mutations resulting from the mutagenic bypass of CPDs formed at TT, TC, and CC sites in DNA (17, 18).Steady-state kinetic analyses...

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“…The log reduction of MS2 was calculated as log reduction ϭ log͑C 0͒ Ϫ log͑C͒ (4) where log(C 0 ) is the log concentration of the unexposed sample obtained from the regression analysis and log(C) is the measured log concentration.…”

Section: Methodsmentioning

confidence: 99%

“…UV light induces damage to the genomes of bacteria, protozoa, and viruses, breaking bonds and forming photodimeric lesions in nucleic acids, DNA, and RNA (1,2). These lesions prevent both transcription and replication and ultimately lead to inactivation of the microorganisms (3,4). Direct UV damage to nucleic acids occurs at the wavelengths absorbed by DNA and RNA, in the germicidal UV region between 200 and 300 nm (5,6).…”

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confidence: 99%

Comparison of UV-Induced Inactivation and RNA Damage in MS2 Phage across the Germicidal UV Spectrum

Beck

Rodríguez

Hawkins

et al. 2016

Appl Environ Microbiol

151

123

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c Polychromatic UV irradiation is a common method of pathogen inactivation in the water treatment industry. To improve its disinfection efficacy, more information on the mechanisms of UV inactivation on microorganisms at wavelengths throughout the germicidal UV spectrum, particularly at below 240 nm, is necessary. This work examined UV inactivation of bacteriophage MS2, a common surrogate for enteric pathogens, as a function of wavelength. The bacteriophage was exposed to monochromatic UV irradiation from a tunable laser at wavelengths of between 210 nm and 290 nm. To evaluate the mechanisms of UV inactivation throughout this wavelength range, RT-qPCR (reverse transcription-quantitative PCR) was performed to measure genomic damage for comparison with genomic damage at 253.7 nm. The results indicate that the rates of RNA damage closely mirror the loss of viral infectivity across the germicidal UV spectrum. This demonstrates that genomic damage is the dominant cause of MS2 inactivation from exposure to germicidal UV irradiation. These findings contrast those for adenovirus, for which MS2 is used as a viral surrogate for validating polychromatic UV reactors. UV irradiation is a common method of disinfection in the water treatment industry. UV light induces damage to the genomes of bacteria, protozoa, and viruses, breaking bonds and forming photodimeric lesions in nucleic acids, DNA, and RNA (1, 2). These lesions prevent both transcription and replication and ultimately lead to inactivation of the microorganisms (3, 4). Direct UV damage to nucleic acids occurs at the wavelengths absorbed by DNA and RNA, in the germicidal UV region between 200 and 300 nm (5, 6). In this wavelength range, however, UV light also damages other cellular and viral components, causing, for example, photochemical reactions in proteins and enzymes (7,8). For this reason, UV sources that emit polychromatic light, across the germicidal UV spectrum, are considered more effective at inactivating certain pathogens than sources that emit monochromatic light at 253.7 nm (9-12). As polychromatic sources become more common, more research is being undertaken to understand the mechanisms of inactivation occurring in pathogens exposed to polychromatic UV irradiation.Male-specific (MS2) coliphage is a single-stranded RNA virus. It infects strains of Escherichia coli that produce F ϩ pili, which serve as viral receptors. The virion consists of a short singlestranded RNA genome (3,569 bases) surrounded by an icosahedral protein capsid, 27 nm in diameter (13). MS2 is commonly used in the water treatment industry as a surrogate for enteroviruses because of its similar size, shape, and genome composition (9,14). It serves as a biodosimeter for UV disinfection studies (15) and for UV reactor validation in North America (14, 16). For reactor validation, MS2 is also used as a surrogate for Cryptosporidium and adenovirus, despite the differences in UV sensitivity and spectral sensitivity between these microorganisms. Recent interest has grown regarding microbial ...

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The Ability of a Variety of Polymerases to Synthesize Past Site-specific cis-syn, trans-syn-II, (6–4), and Dewar Photoproducts of Thymidylyl-(3′→5′)-thymidine

Cited by 61 publications

References 95 publications

Involvement of DnaE, the Second Replicative DNA Polymerase from Bacillus subtilis, in DNA Mutagenesis

Involvement of DnaE, the Second Replicative DNA Polymerase from Bacillus subtilis, in DNA Mutagenesis

Distinct mechanisms of cis-syn thymine dimer bypass by Dpo4 and DNA polymerase η

Comparison of UV-Induced Inactivation and RNA Damage in MS2 Phage across the Germicidal UV Spectrum

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