DNA and Chromosomes
Ada protein– and sequence context–dependent mutagenesis of alkyl phosphotriester lesions in Escherichia coli cellsAlkyl phosphotriester (alkyl-PTE) lesions are frequently induced in DNA and are resistant to repair. Here, we synthesized and characterized methyl (Me)- and n-butyl (nBu)-PTEs in two diastereomeric configurations (Sp and Rp) at six different flanking dinucleotide sites, i.e. XT and TX (X = A, C, or G), and assessed how these lesions impact DNA replication in Escherichia coli cells. When single-stranded vectors contained an Sp-Me-PTE in the sequence contexts of 5′-AT-3′, 5′-CT-3′, or 5′-GT-3′, DNA replication was highly efficient and the replication products for all three sequence contexts contained 85–90% AT and 5–10% TG.
The roles of polymerases ν and θ in replicative bypass of O6- and N2-alkyl-2′-deoxyguanosine lesions in human cellsExogenous and endogenous chemicals can react with DNA to produce DNA lesions that may block DNA replication. Not much is known about the roles of polymerase (Pol) ν and Pol θ in translesion synthesis (TLS) in cells. Here we examined the functions of these two polymerases in bypassing major-groove O6-alkyl-2′-deoxyguanosine (O6-alkyl-dG) and minor-groove N2-alkyl-dG lesions in human cells, where the alkyl groups are ethyl, n-butyl (nBu), and, for O6-alkyl-dG, pyridyloxobutyl. We found that Pol ν and Pol θ promote TLS across major-groove O6-alkyl-dG lesions.
Repair and translesion synthesis of O6-alkylguanine DNA lesions in human cellsO6-alkyl-2′-deoxyguanosine (O6-alkyl-dG) lesions are among the most mutagenic and prevalent alkylated DNA lesions that are associated with cancer initiation and progression. In this study, using a shuttle vector–based strand-specific PCR-competitive replication and adduct bypass assay in conjunction with tandem MS for product identification, we systematically assessed the repair and replicative bypass of a series of O6-alkyl-dG lesions, with the alkyl group being a Me, Et, nPr, iPr, nBu, iBu, or sBu, in several human cell lines.
Human DNA polymerase η has reverse transcriptase activity in cellular environmentsClassical DNA and RNA polymerase (pol) enzymes have defined roles with their respective substrates, but several pols have been found to have multiple functions. We reported previously that purified human DNA pol η (hpol η) can incorporate both deoxyribonucleoside triphosphates (dNTPs) and ribonucleoside triphosphates (rNTPs) and can use both DNA and RNA as substrates. X-ray crystal structures revealed that two pol η residues, Phe-18 and Tyr-92, behave as steric gates to influence sugar selectivity.
DNA replication studies of N-nitroso compound–induced O6-alkyl-2′-deoxyguanosine lesions in Escherichia coliN-Nitroso compounds (NOCs) are common DNA-alkylating agents, are abundantly present in food and tobacco, and can also be generated endogenously. Metabolic activation of some NOCs can give rise to carboxymethylation and pyridyloxobutylation/pyridylhydroxybutylation of DNA, which are known to be carcinogenic and can lead to gastrointestinal and lung cancer, respectively. Herein, using the competitive replication and adduct bypass (CRAB) assay, along with MS- and NMR-based approaches, we assessed the cytotoxic and mutagenic properties of three O6-alkyl-2′-deoxyguanosine (O6-alkyl-dG) adducts, i.e.
Cytotoxic and mutagenic properties of O6-alkyl-2′-deoxyguanosine lesions in Escherichia coli cellsEnvironmental exposure and cellular metabolism can give rise to DNA alkylation, which can occur on the nitrogen and oxygen atoms of nucleobases, as well as on the phosphate backbone. Although O6-alkyl-2′-deoxyguanosine (O6-alkyl-dG) lesions are known to be associated with cancer, not much is known about how the alkyl group structures in these lesions affect their repair and replicative bypass in vivo or how translesion synthesis DNA polymerases influence the latter process. To answer these questions, here we synthesized oligodeoxyribonucleotides harboring seven O6-alkyl-dG lesions, with the alkyl group being Me, Et, nPr, iPr, nBu, iBu, or sBu, and examined the impact of these lesions on DNA replication in Escherichia coli cells.
Cytotoxic and mutagenic properties of minor-groove O2-alkylthymidine lesions in human cellsEndogenous metabolism, environmental exposure, and cancer chemotherapy can lead to alkylation of DNA. It has been well documented that, among the different DNA alkylation products, minor-groove O2-alkylthymidine (O2-alkyldT) lesions are inefficiently repaired. In the present study, we examined how seven O2-alkyldT lesions, with the alkyl group being a Me, Et, nPr, iPr, nBu, iBu, or sBu, are recognized by the DNA replication machinery in human cells. We found that the replication bypass efficiencies of these lesions decrease with increasing length of the alkyl chain, and that these lesions induce substantial frequencies of T→A and T→G mutations.
Impact of tobacco-specific nitrosamine–derived DNA adducts on the efficiency and fidelity of DNA replication in human cellsThe tobacco-derived nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN) are known human carcinogens. Following metabolic activation, NNK and NNN can induce a number of DNA lesions, including several 4-(3-pyridyl)-4-oxobut-1-yl (POB) adducts. However, it remains unclear to what extent these lesions affect the efficiency and accuracy of DNA replication and how their replicative bypass is influenced by translesion synthesis (TLS) DNA polymerases. In this study, we investigated the effects of three stable POB DNA adducts (O2-POB-dT, O4-POB-dT, and O6-POB-dG) on the efficiency and fidelity of DNA replication in HEK293T human cells.
Cross-talk between the H3K36me3 and H4K16ac histone epigenetic marks in DNA double-strand break repairPost-translational modifications of histone proteins regulate numerous cellular processes. Among these modifications, trimethylation of lysine 36 in histone H3 (H3K36me3) and acetylation of lysine 16 in histone H4 (H4K16ac) have important roles in transcriptional regulation and DNA damage response signaling. However, whether these two epigenetic histone marks are mechanistically linked remains unclear. Here we discovered a new pathway through which H3K36me3 stimulates H4K16ac upon DNA double-strand break (DSB) induction in human cells.
Posttranslational Regulation of Human DNA Polymerase ιBackground: Many proteins are subject to posttranslational regulation, such as ubiquitination.Results: Human DNA polymerase ι (polι) can be monoubiquitinated at >27 unique sites, and exposure to naphthoquinones results in polyubiquitination of polι.Conclusion: Ubiquitination sites are located across the entire polι polypeptide as well as various structural motifs.Significance: Ubiquitination at these sites is likely to alter cellular functions of polι in vivo.