Mutation Research/DNA Repair最新文献

5-Formyluracil is a major oxidation product of thymine, formed in DNA in yields comparable to that of 8-oxo-7,8-dihydroguanine by exposure to γ-irradiation. Whereas the repair pathways for removal and the biological effects of persisting 8-oxo-7,8-dihydroguanine are much elucidated, much less attention has been paid to the cellular implications of 5-formyluracil in DNA. Here we review the present state of knowledge in this important area within research on oxidative DNA damage.

XPA is a central protein component of nucleotide excision repair (NER), a ubiquitous, multi-component cellular pathway responsible for the removal and repair of many structurally distinct DNA lesions from the eukaryotic genome. The solution structure of the minimal DNA-binding domain of XPA (XPA-MBD: M98-F219) has recently been determined and chemical shift mapping experiments with ${}^{15}\text{N}$-labeled XPA-MBD show that XPA binds DNA along a basic surface located in the C-terminal loop-rich subdomain. Here, XPA–DNA interactions are further characterized using an XPA fragment containing the minimal DNA-binding domain plus the ERCC1-binding region (XPA-EM: M59-F219). The ${}^{15}\text{N}\text{/}{}^1\text{H}$ HSQC spectrum of XPA-EM closely maps onto the ${}^{15}\text{N}\text{/}{}^1\text{H}$ HSQC spectrum of XPA-MBD, suggesting the DNA-binding domain is intact in the larger XPA fragment. Such a conclusion is corroborated by chemical shift mapping experiments of XPA-EM with a single strand DNA oligomer, dCCAATAACC (d9), that show the same set of ${}^{15}\text{N/}{}^1\text{H}$ HSQC cross peaks are effected by the addition of DNA. However, relative to DNA-free XPA-MBD, the ${}^{15}\text{N/}{}^1\text{H}$ HSQC cross peaks of many of the basic residues in the loop-rich subdomain of DNA-free XPA-EM are less intense, or gone altogether, suggesting the acidic ERRC1-binding region of XPA-EM may associate transiently with the basic DNA-binding surface. While the DNA-binding domain in XPA-EM is structured and functional, ${}^{15}\text{N}$-edited NOESY spectra of XPA-EM indicate that the acidic ERRC1-binding region is unstructured. If the structural features observed for XPA-EM persist in XPA, transient intramolecular association of the ERCC1-binding domain with the DNA-binding region may play a role in the sequential assembly of the NER components.

8-Hydroxyguanine (oh⁸G) is a major form of oxidative DNA damage produced by reactive oxygen species (ROS). The human OGG1 gene encodes a DNA glycosylase that excises oh⁸G from double-stranded DNA. In this study, we investigated a mode of interaction between OGG1 and APEX proteins in the repair of oh⁸G under oxidative stresses. DNA cleavage assay using oh⁸G-containing oligonucleotides showed that the phosphodiester bond on the 3′-side of oh⁸G was cleaved by the AP lyase activity of GST-OGG1 protein and the phosphodiester bond on the 5′-side of oh⁸G was cleaved by the DNA 3′-repair diesterase activity of APEX protein. Gel mobility shift assay showed that the complex of GST-OGG1 protein and oh⁸G-containing oligonucleotides mostly changed into the complex of APEX protein and oligonucleotides by addition of APEX protein into the reaction mixture. We next analyzed alterations in the amount of 8-hydroxydeoxyguanosine (oh⁸dG) in DNA and the levels of OGG1 and APEX expression in HeLa S3 cells treated with 2 mM hypochlorous acid, a kind of ROS. An approximately four-fold increase in the amount of oh⁸G was detected by the HPLC–ECD method. Reverse transcriptase–polymerase chain reaction (RT–PCR) and Western blot analyses indicated that the level of APEX expression increased approximately four-fold, whereas the level of OGG1 expression was unchanged. However, in the DNA cleavage assay, the AP lyase activity of GST-OGG1 protein was significantly increased in the presence of a molar excess of APEX protein. These results indicate that, under severe oxidative stresses, OGG1 mRNA is not induced and the amount of OGG1 protein is not remarkably increased, but the activity of OGG1 protein is enhanced by the increase of APEX protein in the cells.

Mitochondrial DNA integrity is ensured by several nuclear-encoded proteins in vertebrates, and a number of mtDNA alterations in human diseases, including deletions and duplications, have been suspected to result from errors in the mitochondrial recombination pathway. However, the presence of the latter system is still a matter of controversy as RecA proteins display various functions in vitro. In Escherichia coli, RecA plays a central role in homologous recombination by pairing and transferring a single strand to a homologous duplex DNA. To address indirectly the issue of a mitochondrial recombination pathway in vivo, we have constructed a chimeric gene containing an N terminal mitochondrial targeting sequence and the E. coli RecA gene. Cells were transfected by the recombinant plasmid, then tested for their mtDNA repair upon bleomycin treatment. We found an increased repair rate of the mitochondrial DNA in cells expressing RecA as compared to control cells. These results indicate that the transfected cells display an improved mtDNA repair replication pathway due to the exogeneous RecA, likely in synergy with an endogeneous rate-limiting mitochondrial recombination pathway.

Recombination-deficient strains have been proven useful for the understanding of the genetic control of homologous recombination. As the genetic screens used to isolate recombination-deficient (rec⁻) yeast mutants have not been saturated, we sought to develop a simple colony color assay to identify mutants with low or elevated rates of recombination. Using this system we isolated a collection of rec⁻ mutants. We report the characterization of the REC41 gene identified in this way. REC41 is required for normal levels of interplasmid recombination and γ-ray induced mitotic interchromosomal recombination. The rec41-1 mutant failed to grow at 37°C. Microscopic analysis of plated cells showed that 45–50% of them did not form visible colonies at permissive temperature. Haploid cells of the rec41 mutant show the same γ-ray sensitivity as wild type ones. However, the diploid rec41 mutant shows γ-ray sensitivity which is comparable with heterozygous REC41/rec41-1 diploid cells. This fact indicates semidominance of the rec41-1 mutation. Diploid strains homozygous for the rec41 rad52 mutations had the same γ-ray sensitivity as single rad52 diploids and exhibited dramatically decreased growth rate. The expression of the HO gene does not lead to inviability of rec41 cells. The rec41 mutation has an effect on meiosis, likely meiotic recombination, even in the heterozygous state. We cloned the REC41 gene. Sequence analysis revealed that the REC41 gene is encoded by ORF YDR245w. Earlier, this ORF was attributed to MNN10, BED1, SLC2, CAX5 genes. Two multicopy plasmids with suppressers of the rec41-1 mutation (pm21 and pm32) were isolated. The deletion analysis showed that only DNA fragments with the CDC43 and HAC1 genes can partially complement the rec41-1 mutation.

We sequenced a gene encoding AP endonuclease DdAPN in Dictyostelium discoideum. The sequence predicts a protein of 542 amino acids, showing high homology to Escherichia coli Endonuclease IV (Endo IV). There is 45% identity to Endo IV using the C-terminal 282 amino acids of the Dictyostelium protein. The DdAPN conserves nine residues for the metal-binding identified in Endo IV. The truncated DdAPN protein containing these sites partially complemented E. coli RPC501 (xth⁻, nfo⁻).

Resistance of tumors to drugs such as cisplatin and mitomycin C (MMC) is an important factor limiting their usefulness in cancer chemotherapy. The antitumor effects of these drugs are due to the formation of bifunctional adducts in DNA, with cisplatin causing predominantly intrastrand-crosslinks and MMC causing interstrand-crosslinks. The SOS chromotest was used to study the cellular mechanisms that process DNA damage in Escherichia coli exposed to cisplatin, ultraviolet irradiation (UV) and MMC and subsequently facilitate the production of a molecular signal for induction of the SOS response. Strains used in the SOS chromotest have a fusion of lacZ with the sfiA (sulA) gene so that the amount of SOS inducing signal, which is modulated by the ability of the cell to repair DNA, is measured by assaying β-galactosidase activity. SOS induction in a strain proficient in homologous recombination (HR) was compared with that in isogenic strains deficient in HR due to a blocked RecBC pathway caused by a recB mutation or a blocked RecFOR pathway caused by a recO mutation. The effect of cisplatin treatment in a uvrA mutant strain blocked at the first step of NER was compared with that in an isogenic strain proficient in NER. Cellular resistance was measured as percent colony forming units (cfu) for cells treated with increasing doses of cisplatin, MMC and UV relative to that in untreated control cultures. The importance of both HR pathways for resistance to these treatments was demonstrated by decreased survival in mutants with the recB mutant being more sensitive than the recO mutant. SOS induction levels were elevated in the sensitive recB strain relative to the HR proficient strain possibly due to stalled and/or distorted replication forks at crosslinks in DNA. In contrast, induction of SOS was dependent on RecFOR activity that is thought to act at daughter strand gaps in newly synthesized DNA to mediate production of the signal for SOS induction. Proficiency in NER was necessary for both survival and high levels of SOS induction in cisplatin treated cells.

We have previously shown that human cancer cells deficient in DNA mismatch repair (MMR) are resistant to the chemotherapeutic methylating agent temozolomide (TMZ) and can be sensitized by the base excision repair (BER) blocking agent methoxyamine (MX) [21]. To further characterize BER-mediated repair responses to methylating agent-induced DNA damage, we have now evaluated the effect of MX on TMZ-induced DNA single strand breaks (SSB) by alkaline elution and DNA double strand breaks (DSB) by pulsed field gel electrophoresis in SW480 (O⁶-alkylguanine-DNA-alkyltransferase [AGT]+, MMR wild type) and HCT116 (AGT+, MMR deficient) colon cancer cells. SSB were evident in both cell lines after a 2-h exposure to equitoxic doses of temozolomide. MX significantly increased the number of TMZ-induced DNA-SSB in both cell lines. In contrast to SSB, TMZ-induced DNA-DSB were dependent on MMR status and were time-dependent. Levels of 50 kb double stranded DNA fragments in MMR proficient cells were increased after TMZ alone or in combination with O⁶-benzylguanine or MX, whereas, in MMR deficient HCT116 cells, only TMZ plus MX produced significant levels of DNA-DSB. Levels of AP endonuclease, XRCC1 and polymerase β were present in both cell lines and were not significantly altered after MX and TMZ. However, cleavage of a 30-mer double strand substrate by SW480 and HCT116 crude cell extracts was inhibited by MX plus TMZ. Thus, MX potentiation of TMZ cytotoxicity may be explained by the persistence of apurinic/apyrimidinic (AP) sites not further processed due to the presence of MX. Furthermore, in MMR-deficient, TMZ-resistant HCT116 colon cancer cells, MX potentiates TMZ cytotoxicity through formation of large DS-DNA fragmentation and subsequent apoptotic signalling.

We have shown previously that induction of the SOS response is required for efficient nucleotide excision repair (NER) of the major ultraviolet light (UV) induced DNA lesion, the cyclobutane pyrimidine dimer (CPD), but not for repair of 6-4 photoproducts (6-4PP) or for transcription-coupled repair of CPDs [1]. We have proposed that the upregulation of cellular NER capacity occurs in the early stages of the SOS response and enhances the rate of repair of the abundant yet poorly recognized genomic CPDs. The expression of three NER genes, uvrA, uvrB, and uvrD, is upregulated as part of the SOS response. UvrD differs from the others in that it is not involved in lesion recognition but rather in promoting the post-incision steps of NER, including turnover of the UvrBC incision complex. Since uvrC is not induced during the SOS response, its turnover would seem to be of great importance in promoting efficient NER. Here we show that the constitutive level of UvrD is adequate for carrying out efficient NER of both CPDs and 6-4PPs. Thus, the upregulation of uvrA and uvrB genes during the SOS response is sufficient for inducible NER of CPDs. We also show that cells with a limited NER capacity, in this case due to deletion of the uvrD gene, repair 6-4PPs but cannot perform transcription-coupled repair of CPDs, indicating that the 6-4PP is a better substrate for NER than is a CPD targeted for transcription-coupled repair.

In the present study, we evaluated the sensitivity of different Escherichia coli strains to Cumene hydroperoxide (CHP) treatment under distinct conditions of Fe²⁺ availability. Our results showed that the pretreatment with an iron chelator (dipyridyl) protects all the tested strains against CHP toxic effects, but it was not sufficient to abolish the CHP induced mutagenesis. On the other hand, simultaneous pretreatment with both dipyridyl and neocuproine (copper chelator) leads to a complete protection against CHP mutagenic effects. Our data suggest the participation of copper ion in the CHP mutagenesis induced in E. coli.