Chanchal K. Malik

Novel genomic island modifies DNA with 7-deazaguanine derivatives

Significance The discovery of a novel modification system that inserts 7-deazaguanine derivatives in DNA, modifications thought until now to occur only in RNA, is an excellent illustration of the power of biological evolution to alter the ultimate function not only of the distinct proteins but also of entire metabolic pathways. The extensive lateral transfer of the gene cluster responsible for this modification highlights its significance as a previously unrecognized foreign DNA defense system that bacteria and phages use to protect their genomes. The characterization of these DNA modification pathways also opens the door to novel tools to manipulate nucleic acids. The discovery of ∼20-kb gene clusters containing a family of paralogs of tRNA guanosine transglycosylase genes, called tgtA5, alongside 7-cyano-7-deazaguanine (preQ0) synthesis and DNA metabolism genes, led to the hypothesis that 7-deazaguanine derivatives are inserted in DNA. This was established by detecting 2’-deoxy-preQ0 and 2’-deoxy-7-amido-7-deazaguanosine in enzymatic hydrolysates of DNA extracted from the pathogenic, Gram-negative bacteria Salmonella enterica serovar Montevideo. These modifications were absent in the closely related S. enterica serovar Typhimurium LT2 and from a mutant of S. Montevideo, each lacking the gene cluster. This led us to rename the genes of the S. Montevideo cluster as dpdA-K for 7-deazapurine in DNA. Similar gene clusters were analyzed in ∼150 phylogenetically diverse bacteria, and the modifications were detected in DNA from other organisms containing these clusters, including Kineococcus radiotolerans, Comamonas testosteroni, and Sphingopyxis alaskensis. Comparative genomic analysis shows that, in Enterobacteriaceae, the cluster is a genomic island integrated at the leuX locus, and the phylogenetic analysis of the TgtA5 family is consistent with widespread horizontal gene transfer. Comparison of transformation efficiencies of modified or unmodified plasmids into isogenic S. Montevideo strains containing or lacking the cluster strongly suggests a restriction–modification role for the cluster in Enterobacteriaceae. Another preQ0 derivative, 2’-deoxy-7-formamidino-7-deazaguanosine, was found in the Escherichia coli bacteriophage 9g, as predicted from the presence of homologs of genes involved in the synthesis of the archaeosine tRNA modification. These results illustrate a deep and unexpected evolutionary connection between DNA and tRNA metabolism.

Kinetic analysis of the bypass of a bulky DNA lesion catalyzed by human Y-family DNA polymerases.

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 (dG(AP)). If not repaired, this DNA lesion is presumably bypassed in vivo by any of human Y-family DNA polymerases kappa (hPolκ), iota (hPolι), eta (hPolη), and Rev1 (hRev1). Our running start assays demonstrated that each of these enzymes was indeed capable of traversing a site-specifically placed dG(AP) on a synthetic DNA template but that hRev1 was stopped after lesion bypass. The time required to bypass 50% of the dG(AP) sites (t(50)(bypass)) encountered by hPolη, hPolκ, and hPolι was determined to be 2.5 s, 4.1 s, and 106.5 s, respectively. The efficiency order of catalyzing translesion synthesis of dG(AP) (hPolη > hPolκ > hPolι ≫ hRev1) is the same as the order for these human Y-family enzymes to elongate undamaged DNA. Although hPolη bypassed dG(AP) efficiently, replication by both hPolκ and hPolι was strongly stalled at the lesion site and at a site immediately downstream from dG(AP). By employing presteady state kinetic methods, a kinetic basis was established for polymerase pausing at these DNA template sites. Besides efficiency of bypass, the fidelity of those low-fidelity polymerases at these pause sites was also significantly decreased. Thus, if the translesion DNA synthesis of dG(AP)in vivo is catalyzed by a human Y-family DNA polymerase, e.g., hPolη, the process is certainly mutagenic.

Mutational analysis of the C8-guanine adduct of the environmental carcinogen 3-nitrobenzanthrone in human cells: critical roles of DNA polymerases η and κ and Rev1 in error-prone translesion synthesis.

3-Nitrobenzanthrone (3-NBA), a potent mutagen and suspected human carcinogen, is a common environmental pollutant. The genotoxicity of 3-NBA has been associated with its ability to form DNA adducts, including N-(2′-deoxyguanosin-8-yl)-3-aminobenzanthrone (C8-dG-ABA). To investigate the molecular mechanism of C8-dG-ABA mutagenesis in human cells, we have replicated a plasmid containing a single C8-dG-ABA in human embryonic kidney 293T (HEK293T) cells, which yielded 14% mutant progeny. The major types of mutations induced by C8-dG-ABA were G → T > G → A > G → C. siRNA knockdown of the translesion synthesis (TLS) DNA polymerases (pols) in HEK293T cells indicated that pol η, pol κ, pol ι, pol ζ, and Rev1 each have a role in replication across this adduct. The extent of TLS was reduced with each pol knockdown, but the largest decrease (of ∼55% reduction) in the level of TLS occurred in cells with knockdown of pol ζ. Pol η and pol κ were considered the major contributors of the mutagenic TLS, because the mutation frequency (MF) decreased by 70%, when these pols were simultaneously knocked down. Rev1 also is important for mutagenesis, as reflected by the 60% reduction in MF upon Rev1 knockdown, but it probably plays a noncatalytic role by physically interacting with the other two Y-family pols. In contrast, pol ζ appeared to be involved in the error-free bypass of the lesion, because MF increased by 60% in pol ζ knockdown cells. These results provide important mechanistic insight into the bypass of the C8-dG-ABA adduct.

Quantitative analysis of the mutagenic potential of 1-aminopyrene-DNA adduct bypass catalyzed by Y-family DNA polymerases

N-(Deoxyguanosin-8-yl)-1-aminopyrene (dG(AP)) is the predominant nitro polyaromatic hydrocarbon product generated from the air pollutant 1-nitropyrene reacting with DNA. Previous studies have shown that dG(AP) induces genetic mutations in bacterial and mammalian cells. One potential source of these mutations is the error-prone bypass of dG(AP) lesions catalyzed by the low-fidelity Y-family DNA polymerases. To provide a comparative analysis of the mutagenic potential of the translesion DNA synthesis (TLS) of dG(AP), we employed short oligonucleotide sequencing assays (SOSAs) with the model Y-family DNA polymerase from Sulfolobus solfataricus, DNA Polymerase IV (Dpo4), and the human Y-family DNA polymerases eta (hPolη), kappa (hPolκ), and iota (hPolι). Relative to undamaged DNA, all four enzymes generated far more mutations (base deletions, insertions, and substitutions) with a DNA template containing a site-specifically placed dG(AP). Opposite dG(AP) and at an immediate downstream template position, the most frequent mutations made by the three human enzymes were base deletions and the most frequent base substitutions were dAs for all enzymes. Based on the SOSA data, Dpo4 was the least error-prone Y-family DNA polymerase among the four enzymes during the TLS of dG(AP). Among the three human Y-family enzymes, hPolκ made the fewest mutations at all template positions except opposite the lesion site. hPolκ was significantly less error-prone than hPolι and hPolη during the extension of dG(AP) bypass products. Interestingly, the most frequent mutations created by hPolι at all template positions were base deletions. Although hRev1, the fourth human Y-family enzyme, could not extend dG(AP) bypass products in our standing start assays, it preferentially incorporated dCTP opposite the bulky lesion. Collectively, these mutagenic profiles suggest that hPolk and hRev1 are the most suitable human Y-family DNA polymerases to perform TLS of dG(AP) in humans.

Structural Basis for Error-Free Bypass of the 5-N-Methylformamidopyrimidine-dG Lesion by Human DNA Polymerase η and Sulfolobus solfataricus P2 Polymerase IV.

N(6)-(2-Deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine (MeFapy-dG) arises from N7-methylation of deoxyguanosine followed by imidazole ring opening. The lesion has been reported to persist in animal tissues. Previous in vitro replication bypass investigations of the MeFapy-dG adduct revealed predominant insertion of C opposite the lesion, dependent on the identity of the DNA polymerase (Pol) and the local sequence context. Here we report crystal structures of ternary Pol·DNA·dNTP complexes between MeFapy-dG-adducted DNA template:primer duplexes and the Y-family polymerases human Pol η and P2 Pol IV (Dpo4) from Sulfolobus solfataricus. The structures of the hPol η and Dpo4 complexes at the insertion and extension stages, respectively, are representative of error-free replication, with MeFapy-dG in the anti conformation and forming Watson-Crick pairs with dCTP or dC.

Error-prone replication bypass of the imidazole ring-opened formamidopyrimidine deoxyguanosine adduct: Mutagenesis of Ring-Fragmented dG

Addition of hydroxyl radicals to the C8 position of 2′‐deoxyguanosine generates an 8‐hydroxyguanyl radical that can be converted into either 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine or N‐(2‐deoxy‐d‐pentofuranosyl)‐N‐(2,6‐diamino‐4‐hydroxy‐5‐formamidopyrimidine) (Fapy‐dG). The Fapy‐dG adduct can adopt different conformations and in particular, can exist in an unnatural α anomeric configuration in addition to canonical β configuration. Previous studies reported that in 5′‐TGN‐3′ sequences, Fapy‐dG predominantly induced G → T transversions in both mammalian cells and Escherichia coli, suggesting that mutations could be formed either via insertion of a dA opposite the 5′ dT due to primer/template misalignment or as result of direct miscoding. To address this question, single‐stranded vectors containing a site‐specific Fapy‐dG adduct were generated to vary the identity of the 5′ nucleotide. Following vector replication in primate cells (COS7), complex mutation spectra were observed that included ∼3–5% G → T transversions and ∼14–21% G → A transitions. There was no correlation apparent between the identity of the 5′ nucleotide and spectra of mutations. When conditions for vector preparation were modified to favor the β anomer, frequencies of both G → T and G → A substitutions were significantly reduced. Mutation frequencies in wild‐type E. coli and a mutant deficient in damage‐inducible DNA polymerases were significantly lower than detected in COS7 and spectra were dominated by deletions. Thus, mutagenic bypass of Fapy‐dG can proceed via mechanisms that are different from the previously proposed primer/template misalignment or direct misinsertions of dA or dT opposite to the β anomer of Fapy‐dG. Environ. Mol. Mutagen. 58:182–189, 2017.

Synthesis of Oligodeoxynucleotides Containing a C8‐2′‐Deoxyguanosine Adduct Formed by the Carcinogen 3‐Nitrobenzanthrone

This unit describes the detailed procedure in five parts for the synthesis of the C8‐2′‐deoxyguanosine‐3‐aminobenzanthrone adduct located in a desired site in an oligonucleotide. The synthesis of the protected 2′‐deoxyguanosine, O 6‐benzyl‐N 2‐DMTr‐3′‐5′‐bisTBDMS‐C8‐Br‐2′‐deoxyguanosine, is described in the first part. The synthesis of the reduced carcinogen 3‐aminobenzanthrone is detailed in part two. The third part outlines the key step of the adduct formation between the reduced carcinogen and the protected nucleoside by a palladium‐catalyzed cross coupling reaction. The final two parts describe phosphoramidite synthesis from the nucleoside‐carcinogen adduct followed by its site‐specific incorporation into DNA by solid‐phase oligonucleotide synthesis. The adducted oligonucleotides are purified by reversed‐phase HPLC and characterized by mass spectrometry.