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This work provides evidence that the product of the RDH54 gene participates in the coordination of some repair pathways of DNA lesions. The unique point mutation rdh54–29 described in our previous works confers the phenotype markedly differing from that of the strain with a full deletion of gene RDH54. The epistatic type of interaction between mutations rdh 54–29 and apn 1Δ allowed the product of gene RDH54 to be attributed to the base excision repair pathway. However, a pleiotropic effect of mutation rdh54–29 manifested as sensitivity to a wide spectrum of DNA-damagi ng agents suggests that Rdh54 is involved in the regulation of several DNA repair pathways. To verify this hypothesis, the direct influence of mutation rdh54–29 on recombination and mutagenesis was evaluated. The results obtained led to the assumption that, in addition to the involvement in base excision repair, Rdh54p may play a certain role in the coordination of DNA lesion repair by various systems, including recombinational and mutagenic repair pathways or nucleotide excision repair. This function supposedly is mediated through modification of chromatin structure at the location of DNA lesion, in particular, by alleviation of DNA-hi stone bonds, thus rendering DNA more susceptible to the action of various repair proteins. [ABSTRACT FROM AUTHOR]

Simple Summary: This review offers a thorough examination of the potential role and structural properties of MGMT, the progression in targeting MGMT, and the interactions between MGMT and DNA repair pathways involved in processing DNA lesions. O6-methylguanine-DNA methyltransferase (MGMT or AGT) is a DNA repair protein with the capability to remove alkyl groups from O6-AlkylG adducts. Moreover, MGMT plays a crucial role in repairing DNA damage induced by methylating agents like temozolomide and chloroethylating agents such as carmustine, and thereby contributes to chemotherapeutic resistance when these agents are used. This review delves into the structural roles and repair mechanisms of MGMT, with emphasis on the potential structural and functional roles of the N-terminal domain of MGMT. It also explores the development of cancer therapeutic strategies that target MGMT. Finally, it discusses the intriguing crosstalk between MGMT and other DNA repair pathways. [ABSTRACT FROM AUTHOR]

DNA alkyltransferase and alkyltransferase-like family proteins are responsible for the repair of highly mutagenic and cytotoxic O6-alkylguanine and O4-alkylthymine bases in DNA. Their mechanism involves binding to the damaged DNA and flipping the base out of the DNA helix into the active site pocket in the protein. Alkyltransferases then directly and irreversibly transfer the alkyl group from the base to the active site cysteine residue. In contrast, alkyltransferase-like proteins recruit nucleotide excision repair components for O6-alkylguanine elimination. One or more of these proteins are found in all kingdoms of life, and where this has been determined, their overall DNA repair mechanism is strictly conserved between organisms. Nevertheless, between species, subtle as well as more extensive differences that affect target lesion preferences and/or introduce additional protein functions have evolved. Examining these differences and their functional consequences is intricately entwined with understanding the details of their DNA repair mechanism(s) and their biological roles. In this review, we will present and discuss various aspects of the current status of knowledge on this intriguing protein family. [ABSTRACT FROM AUTHOR]

Bacteria use various strategies to become antibiotic resistant. The molecular details of these strategies are not fully understood. We can increase our understanding by investigating the same strategies found in antibiotic‐producing bacteria. In this work, we characterize the self‐resistance protein Ecm16 encoded by echinomycin‐producing bacteria. Ecm16 is a structural homolog of the nucleotide excision repair protein UvrA. Expression of ecm16 in the heterologous system Escherichia coli was sufficient to render resistance against echinomycin. Ecm16 binds DNA (double‐stranded and single‐stranded) using a nucleotide‐independent binding mode. Ecm16's binding affinity for DNA increased by 1.7‐fold when the DNA is intercalated with echinomycin. Ecm16 can render resistance against echinomycin toxicity independently of the nucleotide excision repair system. Similar to UvrA, Ecm16 has ATPase activity, and this activity is essential for Ecm16's ability to render echinomycin resistance. Notably, UvrA and Ecm16 were unable to complement each other's function. Together, our findings identify new mechanistic details of how a refurbished DNA repair protein Ecm16 can specifically render resistance to the DNA intercalator echinomycin. [ABSTRACT FROM AUTHOR]

During meiosis, homologous chromosomes must recognize, pair, and recombine with one another to ensure the formation of inter-homologue crossover events, which, together with sister chromatid cohesion, promote correct chromosome orientation on the first meiotic spindle. Crossover formation requires the assembly of axial elements, proteinaceous structures that assemble along the length of each chromosome during early meiosis, as well as checkpoint mechanisms that control meiotic progression by monitoring pairing and recombination intermediates. A conserved family of proteins defined by the presence of a HORMA (HOp1, Rev7, MAd2) domain, referred to as HORMADs, associate with axial elements to control key events of meiotic prophase. The highly conserved HORMA domain comprises a flexible safety belt sequence, enabling it to adopt at least two of the following protein conformations: one closed, where the safety belt encircles a small peptide motif present within an interacting protein, causing its topological entrapment, and the other open, where the safety belt is reorganized and no interactor is trapped. Although functional studies in multiple organisms have revealed that HORMADs are crucial regulators of meiosis, the mechanisms by which HORMADs implement key meiotic events remain poorly understood. In this review, we summarize protein complexes formed by HORMADs, discuss their roles during meiosis in different organisms, draw comparisons to better characterize non-meiotic HORMADs (MAD2 and REV7), and highlight possible areas for future research. [ABSTRACT FROM AUTHOR]

When reproduction in fungi takes place by sexual means, meiosis enables the formation of haploid spores from diploid precursor cells. Copper is required for completion of meiosis in Schizosaccharomyces pombe. During the meiotic program, genes encoding copper transporters exhibit distinct temporal expression profiles. In the case of the major facilitator copper transporter 1 (Mfc1), its maximal expression is induced during middle-phase meiosis and requires the presence of the Zn6Cys2 binuclear cluster-type transcription factor Mca1. In this study, we further characterize the mechanism by which Mca1 affects the copper-starvation-induced expression of mfc1+. Using a chromatin immunoprecipitation (ChIP) approach, results showed that a functional Mca1-TAP occupies the mfc1+ promoter irrespective of whether this gene is transcriptionally active. Under conditions of copper starvation, results showed that the presence of Mca1 promotes RNA polymerase II (Pol II) occupancy along the mfc1+ transcribed region. In contrast, Pol II did not significantly occupy the mfc1+ locus in meiotic cells that were incubated in the presence of copper. Further analysis by ChIP assays revealed that binding of Pol II to chromatin at the chromosomal locus of mfc1+ is exclusively detected during meiosis and absent in cells proliferating in mitosis. Protein function analysis of a series of internal mutants compared to the full-length Mca1 identified a minimal form of Mca1 consisting of its DNA-binding domain (residues 1 to 150) fused to the amino acids 299 to 600. This shorter form is sufficient to enhance Pol II occupancy at the mfc1+ locus under low copper conditions. Taken together, these results revealed novel characteristics of Mca1 and identified an internal region of Mca1 that is required to promote Pol II-dependent mfc1+ transcription during meiosis. [ABSTRACT FROM AUTHOR]

To identify new proteins required for faithful meiotic chromosome segregation, we screened a Schizosaccharomyces pombe deletion mutant library and found that deletion of the dbl2 gene led to missegregation of chromosomes during meiosis. Analyses of both live and fixed cells showed that dbl2Δ mutant cells frequently failed to segregate homologous chromosomes to opposite poles during meiosis I. Removing Rec12 (Spo11 homolog) to eliminate meiotic DNA double-strand breaks (DSBs) suppressed the segregation defect in dbl2Δ cells, indicating that Dbl2 acts after the initiation of meiotic recombination. Analyses of DSBs and Holliday junctions revealed no significant defect in their formation or processing in dbl2Δ mutant cells, although some Rec12-dependent DNA joint molecules persisted late in meiosis. Failure to segregate chromosomes in the absence of Dbl2 correlated with persistent Rad51 foci, and deletion of rad51 or genes encoding Rad51 mediators also suppressed the segregation defect of dbl2Δ. Formation of foci of Fbh1, an F-box helicase that efficiently dismantles Rad51-DNA filaments, was impaired in dbl2Δ cells. Our results suggest that Dbl2 is a novel regulator of Fbh1 and thereby Rad51-dependent DSB repair required for proper meiotic chromosome segregation and viable sex cell formation. The wide conservation of these proteins suggests that our results apply to many species. [ABSTRACT FROM AUTHOR]

The fine conformational subtleties of DNA structure modulate many fundamental cellular processes including gene activation/repression, cellular division, and DNA repair. Most of these cellular processes rely on the conformational heterogeneity of specific DNA sequences. Factors including those structural characteristics inherent in the particular base sequence as well as those induced through interaction with solvent components combine to produce fine DNA structural variation including helical flexibility and conformation. Cation-pi interactions between solvent cations or their first hydration shell waters and the faces of DNA bases form sequence selectively and contribute to DNA structural heterogeneity. In this paper, we detect and characterize the binding patterns found in cation-pi interactions between solvent cations and DNA bases in a set of high resolution x-ray crystal structures. Specifically, we found that monovalent cations (Tl+) and the polarized first hydration shell waters of divalent cations (Mg2+, Ca2+) form cation-pi interactions with DNA bases stabilizing unstacked conformations. When these cation-pi interactions are combined with electrostatic interactions a pattern of specific binding motifs is formed within the grooves. [ABSTRACT FROM AUTHOR]

O 6-(carboxymethyl)guanine (O 6-CMG) and O 6-(4-oxo-4-(3-pyridyl)butyl)guanine (O 6-pobG) are toxic lesions formed in DNA following exposure to alkylating agents. O 6-CMG results from exposure to nitrosated glycine or nitrosated bile acid conjugates and may be associated with diets rich in red meat. O 6-pobG lesions are derived from alkylating agents found in tobacco smoke. Efficient syntheses of oligodeoxyribonucleotides (ODNs) containing O 6-CMG and O 6-pobG are described that involve nucleophilic displacement by the appropriate alcohol on a common synthetic ODN containing the reactive base 2-amino-6-methylsulfonylpurine. ODNs containing O 6-pobG and O 6-CMG were found to be good substrates for the S. pombe alkyltransferase-like protein Atl1. [Supplemental materials are available for this article. Go to the publisher's online edition of Nucleosides, Nucleotides & Nucleic Acids to view the free supplemental file.] [ABSTRACT FROM PUBLISHER]

Alkylation is a type of stress that is fatal to cells. However, cells have various responses to alkylation. Alkyltransferase-like (ATL) protein is a novel protein involved in the repair of alkylated DNA; however, its repair mechanism at the molecular level is unclear. DNA microarray analysis revealed that the upregulation of 71 genes because of treatment with an alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine was related to the presence of TTHA1564, the ATL protein from Thermus thermophilus HB8. Affinity chromatography showed a direct interaction of purified TTHA1564 with purified RNA polymerase holoenzyme. The amino acid sequence of TTHA1564 is homologous to that of the C-terminal domain of Ada protein, which acts as a transcriptional activator. These results suggest that TTHA1564 might act as a transcriptional regulator. The results of DNA microarray analysis also implied that the alkylating agent induced oxidation stress in addition to alkylation stress. [ABSTRACT FROM PUBLISHER]

In fission yeast, meiotic prophase nuclei develop structures known as linear elements (LinEs), instead of a canonical synaptonemal complex. LinEs contain Rec10 protein. While Rec10 is essential for meiotic recombination, the precise role of LinEs in this process is unknown. Using in situ immunostaining, we show that Rec7 (which is required for meiosis-specific DNA double-strand break (DSB) formation) aggregates in foci on LinEs. The strand exchange protein Rad51, which is known to mark the sites of DSBs, also localizes to LinEs, although to a lesser degree. The number of Rec7 foci corresponds well with the average number of genetic recombination events per meiosis suggesting that Rec7 marks the sites of recombination. Rec7 and Rad51 foci do not co-localize, presumably because they act sequentially on recombination sites. The localization of Rec7 is dependent on Rec10 but independent of the DSB-inducing protein Rec12/Spo11. Neither Rec7 nor Rad51 localization depends on the LinE-associated proteins Hop1 and Mek1, but the formation of Rad51 foci depends on Rec10, Rec7, and, as expected, Rec12/Spo11. We propose that LinEs form around designated recombination sites before the induction of DSBs and that most, if not all, meiotic recombination initiates within the setting provided by LinEs. [ABSTRACT FROM AUTHOR]