Abstract:1-Nitropyrene (1-NP), a mutagen and potential carcinogen, is the most abundant nitro polyaromatic hydrocarbon in diesel exhaust, which reacts with DNA to form predominantly N-(deoxyguanosin-8-yl)-1-aminopyrene (dGAP). If not repaired, this DNA lesion is presumably bypassed in vivo by any of human Y-family DNA polymerases kappa (hPolκ), iota (hPolτ), eta (hPolη), and Rev1 (hRev1). Our running start assays demonstrated that each of these enzymes was indeed capable of traversing a site-specifically placed dGAP on… Show more
“…The base substitution rate for hPolη replicating the undamaged DNA template was calculated to be 2.8 × 10 − 2 by using HT-SOSA. This value is comparable with the dNTP misincorporation fidelity of 5.6 × 10 − 2 , as measured by steady-state kinetic assays (30), and 2.0 × 10 − 3 to 2.1 × 10 − 2 , as measured by pre-steady-state kinetic assays (31), validating HT-SOSA as an effective method to determine the error rates of lesion bypass. Figure 1.Comparison of the preferred actions of human Y-family DNA polymerases opposite a cis–syn TT-dimer.…”
Cellular genomes are constantly damaged by endogenous and exogenous agents that covalently and structurally modify DNA to produce DNA lesions. Although most lesions are mended by various DNA repair pathways in vivo, a significant number of damage sites persist during genomic replication. Our understanding of the mutagenic outcomes derived from these unrepaired DNA lesions has been hindered by the low throughput of existing sequencing methods. Therefore, we have developed a cost-effective high-throughput short oligonucleotide sequencing assay that uses next-generation DNA sequencing technology for the assessment of the mutagenic profiles of translesion DNA synthesis catalyzed by any error-prone DNA polymerase. The vast amount of sequencing data produced were aligned and quantified by using our novel software. As an example, the high-throughput short oligonucleotide sequencing assay was used to analyze the types and frequencies of mutations upstream, downstream and at a site-specifically placed cis–syn thymidine–thymidine dimer generated individually by three lesion-bypass human Y-family DNA polymerases.
“…The base substitution rate for hPolη replicating the undamaged DNA template was calculated to be 2.8 × 10 − 2 by using HT-SOSA. This value is comparable with the dNTP misincorporation fidelity of 5.6 × 10 − 2 , as measured by steady-state kinetic assays (30), and 2.0 × 10 − 3 to 2.1 × 10 − 2 , as measured by pre-steady-state kinetic assays (31), validating HT-SOSA as an effective method to determine the error rates of lesion bypass. Figure 1.Comparison of the preferred actions of human Y-family DNA polymerases opposite a cis–syn TT-dimer.…”
Cellular genomes are constantly damaged by endogenous and exogenous agents that covalently and structurally modify DNA to produce DNA lesions. Although most lesions are mended by various DNA repair pathways in vivo, a significant number of damage sites persist during genomic replication. Our understanding of the mutagenic outcomes derived from these unrepaired DNA lesions has been hindered by the low throughput of existing sequencing methods. Therefore, we have developed a cost-effective high-throughput short oligonucleotide sequencing assay that uses next-generation DNA sequencing technology for the assessment of the mutagenic profiles of translesion DNA synthesis catalyzed by any error-prone DNA polymerase. The vast amount of sequencing data produced were aligned and quantified by using our novel software. As an example, the high-throughput short oligonucleotide sequencing assay was used to analyze the types and frequencies of mutations upstream, downstream and at a site-specifically placed cis–syn thymidine–thymidine dimer generated individually by three lesion-bypass human Y-family DNA polymerases.
Background: DNA polymerase is a low fidelity polymerase with an unknown function. Results: The kinetic mechanism of correct and incorrect base pair formation was determined. Conclusion: Phosphodiester bond formation is rapid for both correct and mispair formation. Significance: The presence of a mispair does not induce the polymerase to adopt a low catalytic conformation.
“…This new mechanism is likely more accurate than the one in Scheme 1A to explain the bypass of an abasic site, 55 a cisplatin-d(GpG) adduct, 56 a dG AP lesion, 41 and a dG C8- N --ABA lesion 57 by Dpo4 and the bypass of a dG AP lesion by human Y-family DNA polymerases. 42 …”
1-Nitropyrene (1-NP), an environmental pollutant, induces DNA damage in vivo and is considered to be carcinogenic. The DNA adducts formed by the 1-NP metabolites stall replicative DNA polymerases but are presumably bypassed by error-prone Y-family DNA polymerases at the expense of replication fidelity and efficiency in vivo. Our running start assays confirmed that a site-specifically placed 8-(deoxyguanosin-N2-yl)-1-aminopyrene (dG1,8), one of the DNA adducts derived from 1-NP, can be bypassed by Sulfolobus solfataricus DNA polymerase IV (Dpo4), although this representative Y-family enzyme was paused strongly by the lesion. Pre-steady-state kinetic assays were employed to determine the low nucleotide incorporation fidelity and establish a minimal kinetic mechanism for the dG1,8 bypass by Dpo4. To reveal a structural basis for dCTP incorporation opposite dG1,8, we solved the crystal structures of the complexes of Dpo4 and DNA containing a templating dG1,8 lesion in the absence or presence of dCTP. The Dpo4·DNA-dG1,8 binary structure shows that the aminopyrene moiety of the lesion stacks against the primer/template junction pair, while its dG moiety projected into the cleft between the Finger and Little Finger domains of Dpo4. In the Dpo4·DNA-dG1,8·dCTP ternary structure, the aminopyrene moiety of the dG1,8 lesion, is sandwiched between the nascent and junction base pairs, while its base is present in the major groove. Moreover, dCTP forms a Watson–Crick base pair with dG, two nucleotides upstream from the dG1,8 site, creating a complex for “-2” frameshift mutation. Mechanistically, these crystal structures provide additional insight into the aforementioned minimal kinetic mechanism.
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