Resistance-guided mining of bacterial genotoxins defines a family of DNA glycosylases

Unique DNA repair enzymes that provide self-resistance against genotoxic natural products have been discovered recently in bacterial biosynthetic gene clusters (BGCs). The DNA glycosylase AlkZ belongs to a superfamily of uncharacterized proteins found in antibiotic producers and pathogens, but despite its importance to azinomycin B resistance, the roles of AlkZ orthologs in production of other natural products are unknown. Here, we analyze the genomic distribution and use a resistance-based genome mining approach to identify Streptomyces AlkZ homologs associated with known and uncharacterized BGCs. We show that the ortholog associated with synthesis of the alkylating agent hedamycin excises hedamycin-DNA adducts and provides resistance to the genotoxin in cells. Our results define AlkZ in self-resistance to specific antimicrobials and implicate a related but distinct homolog, which we name AlkX, in protection against an array of genotoxins. This work provides a framework for targeted discovery of new genotoxic compounds with therapeutic potential.

The interplay of supercoiling and thymine dimers in DNA

Thymine dimers are a major mutagenic photoproduct induced by UV radiation. While they have been the subject of extensive theoretical and experimental investigations, questions of how DNA supercoiling affects local defect properties, or, conversely, how the presence of such defects changes global supercoiled structure, are largely unexplored. Here we introduce a model of thymine dimers in the oxDNA forcefield, and validate it by comparison to melting experiments and structural measurements of the thymine dimer induced bend angle. We performed extensive molecular dynamics simulations of double-stranded DNA as a function of external twist and force. Compared to undamaged DNA, the presence of a thymine dimer lowers the supercoiling densities at which plectonemes and bubbles occur. For biologically relevant supercoiling densities and forces, thymine dimers can preferentially segregate to the tips of the plectonemes, where they enhance the probability of a localized tip-bubble. This mechanism increases the probability of highly bent and denatured states at the thymine dimer site, which may facilitate repair enzyme binding. Thymine dimer-induced tip-bubbles also pin plectonemes, which may help repair enzymes to locate damage. We hypothesize that the interplay of supercoiling and local defects plays an important role for a wider set of DNA damage repair systems.

The Cdc14 Phosphatase Controls Resolution of Recombination Intermediates and Crossover Formation during Meiosis

Meiotic defects derived from incorrect DNA repair during gametogenesis can lead to mutations, aneuploidies and infertility. The coordinated resolution of meiotic recombination intermediates is required for crossover formation, ultimately necessary for the accurate completion of both rounds of chromosome segregation. Numerous master kinases orchestrate the correct assembly and activity of the repair machinery. Although much less is known, the reversal of phosphorylation events in meiosis must also be key to coordinate the timing and functionality of repair enzymes. Cdc14 is a crucial phosphatase required for the dephosphorylation of multiple CDK1 targets in many eukaryotes. Mutations that inactivate this phosphatase lead to meiotic failure, but until now it was unknown if Cdc14 plays a direct role in meiotic recombination. Here, we show that the elimination of Cdc14 leads to severe defects in the processing and resolution of recombination intermediates, causing a drastic depletion in crossovers when other repair pathways are compromised. We also show that Cdc14 is required for the correct activity and localization of the Holliday Junction resolvase Yen1/GEN1. We reveal that Cdc14 regulates Yen1 activity from meiosis I onwards, and this function is essential for crossover resolution in the absence of other repair pathways. We also demonstrate that Cdc14 and Yen1 are required to safeguard sister chromatid segregation during the second meiotic division, a late action that is independent of the earlier role in crossover formation. Thus, this work uncovers previously undescribed functions of the evolutionary conserved Cdc14 phosphatase in the regulation of meiotic recombination.

Sequence Dependent Repair of 1,N6-Ethenoadenine by DNA Repair Enzymes ALKBH2, ALKBH3, and AlkB

Mutation patterns of DNA adducts, such as mutational spectra and signatures, are useful tools for diagnostic and prognostic purposes. Mutational spectra of carcinogens derive from three sources: adduct formation, replication bypass, and repair. Here, we consider the repair aspect of 1,N6-ethenoadenine (εA) by the 2-oxoglutarate/Fe(II)-dependent AlkB family enzymes. Specifically, we investigated εA repair across 16 possible sequence contexts (5′/3′ flanking base to εA varied as G/A/T/C). The results revealed that repair efficiency is altered according to sequence, enzyme, and strand context (ss- versus ds-DNA). The methods can be used to study other aspects of mutational spectra or other pathways of repair.

Insights of Cisplatin Resistance in Cervical Cancer: A Decision Making for Cellular Survival

The clinical scenario of acquired cisplatin resistance is considered as a major impediment in cervical cancer treatment. Bulky drug-DNA adducts formed by cisplatin elicits DNA damage response (DDR) which either subsequently induces apoptosis in the cervical cancer cells or enables them to adapt with drug assault by invigorating pro-survival molecular cascades. When HPV infected cervical cancer cells encounter cisplatin, a complex molecular interaction between deregulated tumor suppressors, DNA damage-repair enzymes, and prosurvival molecules get initiated. Ambiguous molecular triggers allow cancer cells to cull apoptosis by opting for a survival fate. Overriding of the apoptotic cues by the pro-survival cues renders a cisplatin resistant phenotype in the tumor microenvironment. The present review undrapes the impact of deregulated signaling nexus formed due to crosstalk of the key molecules related to cell survival and apoptosis in orchestrating platinum resistance in cervical cancer.