The use of modified and non-natural nucleotides provide unique insights into pro-mutagenic replication catalyzed by polymerase eta

Abstract

This report evaluates the pro-mutagenic behavior of 8-oxo-guanine (8-oxo-G) by quantifying the ability of high-fidelity and specialized DNA polymerases to incorporate natural and modified nucleotides opposite this lesion. Although high-fidelity DNA polymerases such as pol δ and the bacteriophage T4 DNA polymerase replicating 8-oxo-G in an error-prone manner, they display remarkably low efficiencies for TLS compared to normal DNA synthesis. In contrast, pol η shows a combination of high efficiency and low fidelity when replicating 8-oxo-G. These combined properties are consistent with a pro-mutagenic role for pol η when replicating this DNA lesion. Studies using modified nucleotide analogs show that pol η relies heavily on hydrogen-bonding interactions during translesion DNA synthesis. However, nucleobase modifications such as alkylation to the N2 position of guanine significantly increase error-prone synthesis catalyzed by pol η when replicating 8-oxo-G. Molecular modeling studies demonstrate the existence of a hydrophobic pocket in pol η that participates in the increased utilization of certain hydrophobic nucleotides. A model is proposed for enhanced pro-mutagenic replication catalyzed by pol η that couples efficient incorporation of damaged nucleotides opposite oxidized DNA lesions created by reactive oxygen species. The biological implications of this model toward increasing mutagenic events in lung cancer are discussed.

MutT homologue 1 (MTH1) removes N6-methyl-dATP from the dNTP pool

“…Due to the low DNA levels of N6-methyl-dA even a microevent in N6methyl-dA base modification, for example through incorporation of N6-methyl-dATP into the DNA during replication or DNA repair, may have notable consequences for the cell by disturbing the regulation of gene expression. We show through performing in vivo experiments using zebrafish that N6-methyl-dATP is used as a substrate for eukaryotic DNA polymerases during replication and that it is incorporated into DNA, which confirms what previously has been shown in in vitro experiments (41). The significant effects of alterations in DNA levels of N6-methyladenine reported in the litterature suggests that it is likely important for the cell to remove N6methyl-dATP from the nucleotide pool and thereby prevent its incorporation into DNA.…”

Distinctive Formation of a DNA–Protein Cross-Link during the Repair of DNA Oxidative Damage: Insights into Human Disease from MD Simulations and QM/MM Calculations

“…The generation of reactive oxygen species in human cells from endogenous processes (e.g., mitochondrial aerobic metabolism and inflammatory responses) , or exogenous sources (e.g., pollutants, radiation, tobacco smoke, and heavy metals) − has been linked to a range of health issues, including cancer, diabetes, cardiovascular disease, chronic inflammation, neurodegeneration (Alzheimer’s and Parkinson’s diseases), asthma, and male infertility. − These detrimental effects can result from reactive oxygen species (ROS) altering the chemical structure and base-pairing properties of the DNA nucleobases. , For example, 8-oxo-7,8-dihydroguanine (8oG), a major ROS product that has been estimated to arise 1000–7000 times per cell per day, frequently mispairs with adenine during replication, − which ultimately causes G:C → T:A transverse mutations. To prevent these harmful mutations, the human adenine DNA glycosylase homologue (MUTYH) aids the reversal of 8oG:A pairs by catalyzing deglycosylation of 2′-deoxyadenosine (dA) as part of the base excision repair (BER) pathway .…”

Transient activation of tumoral DNA damage tolerance pathway coupled with immune checkpoint blockade exerts durable tumor regression in mouse melanoma

“…The major Y‐family TLS polymerases eta (Kannouche et al, 2004; McIlwraith et al, 2005; Tissier et al, 2004) and kappa (Bavoux et al, 2005; Ogi & Lehmann, 2006) carry out inserter and extender activities, respectively, while interacting with regulatory and scaffold proteins that confer proficiency and specificity (Bienko et al, 2005; Lehmann, 2006; McIntyre & Woodgate, 2015; Sale et al, 2012; Vaisman & Woodgate, 2017). The network of TLS polymerases is tightly regulated, because aside from enabling survival in crisis, TLS polymerases also can increase replication errors (Choi et al, 2016; Ghosal & Chen, 2013; Ling et al, 2004; Vaisman & Woodgate, 2017). While this process is detrimental in normal cells, elevated mutational burden in tumor cells can generate neoantigens and enhance the antitumor immune response (Lauss et al, 2017; Samstein et al, 2019).…”