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Simple sequence repeats (SSRs) are found throughout the genome, and under some conditions can change in length over time. Germline and somatic expansions of trinucleotide repeats are associated with a series of severely disabling illnesses, including Huntington's disease. The underlying mechanisms that effect SSR expansions and contractions have been experimentally elusive, but models suggesting a role for DNA repair have been proposed, in particular the involvement of transcription-coupled nucleotide excision repair (TCNER) that removes transcription-blocking DNA damage from the transcribed strand of actively expressed genes. If the formation of secondary DNA structures that are associated with SSRs were to block RNA polymerase progression, TCNER could be activated, resulting in the removal of the aberrant structure and a concomitant change in the region's length. To test this, TCNER activity in primary human fibroblasts was assessed on defined DNA substrates containing extrahelical DNA loops that lack discernible internal base pairs or DNA stem-loops that contain base pairs within the stem. The results show that both structures impede transcription elongation, but there is no corresponding evidence that nucleotide excision repair (NER) or TCNER operates to remove them.

DNA adducts derived from carcinogenic polycyclic aromatic hydrocarbons like benzo[a]pyrene (B[a]P) and benzo[c]phenanthrene (B[c]Ph) impede replication and transcription, resulting in aberrant cell division and gene expression. Global nucleotide excision repair (NER) and transcription-coupled DNA repair (TCR) are among the DNA repair pathways that evolved to maintain genome integrity by removing DNA damage. The interplay between global NER and TCR in repairing the polycyclic aromatic hydrocarbon-derived DNA adducts (+)-trans-anti-B[a]P-N(6)-dA, which is subject to NER and blocks transcription in vitro, and (+)-trans-anti-B[c]Ph-N(6)-dA, which is a poor substrate for NER but also blocks transcription in vitro, was tested. The results show that both adducts inhibit transcription in human cells that lack both NER and TCR. The (+)-trans-anti-B[a]P-N(6)-dA lesion exhibited no detectable effect on transcription in cells proficient in NER but lacking TCR, indicating that NER can remove the lesion in the absence of TCR, which is consistent with in vitro data. In primary human cells lacking NER, (+)-trans-anti-B[a]P-N(6)-dA exhibited a deleterious effect on transcription that was less severe than in cells lacking both pathways, suggesting that TCR can repair the adduct but not as effectively as global NER. In contrast, (+)-trans-anti-B[c]Ph-N(6)-dA dramatically reduces transcript production in cells proficient in global NER but lacking TCR, indicating that TCR is necessary for the removal of this adduct, which is consistent with in vitro data showing that it is a poor substrate for NER. Hence, both global NER and TCR enhance the recovery of gene expression following DNA damage, and TCR plays an important role in removing DNA damage that is refractory to NER., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

DNA damage located within a gene's transcription unit can cause RNA polymerase to stall at the modified site, resulting in a truncated transcript, or progress past, producing full-length RNA. However, it is not immediately apparent why some lesions pose strong barriers to elongation while others do not. Studies using site-specifically damaged DNA templates have demonstrated that a wide range of lesions can impede the progress of elongating transcription complexes. The collected results of this work provide evidence for the idea that subtle structural elements can influence how an RNA polymerase behaves when it encounters a DNA adduct during elongation. These elements include: (1) the ability of the RNA polymerase active site to accommodate the damaged base; (2) the size and shape of the adduct, which includes the specific modified base; (3) the stereochemistry of the adduct; (4) the base incorporated into the growing transcript; and (5) the local DNA sequence.

Chemical and physical agents can alter the structure of DNA by modifying the bases and the phosphate-sugar backbone, consequently compromising both replication and transcription. During transcription elongation, RNA polymerase complexes can stall at a damaged site in DNA and mark the lesion for repair by a subset of proteins that are utilized to execute nucleotide excision repair, a pathway commonly associated with the removal of bulky DNA damage from the genome. This RNA polymerase-induced repair pathway is called transcription-coupled nucleotide excision repair. Although our understanding of DNA lesion effects on transcription elongation and the associated effects of stalled transcription complexes on DNA repair is broadening, the attainment of critical data is somewhat impeded by labor-intensive, time- consuming processes that are required to prepare damaged DNA templates. Here, we describe an approach for building linear DNA templates that contain a single, site-specific DNA lesion and support transcription by human RNA polymerase II. The method is rapid, making use of biotin-avidin interactions and paramagnetic particles to purify the final product. Data are supplied demonstrating that these templates support transcription, and we emphasize the potential versatility of the protocol and compare it with other published methods.

Many different cellular pathways have evolved to protect the genome from the deleterious effects of DNA damage that result from exposure to chemical and physical agents. Among these is a process called transcription-coupled repair (TCR) that catalyzes the removal of DNA lesions from the transcribed strand of expressed genes, often resulting in a preferential bias of damage clearance from this strand relative to its non-transcribed counterpart. Lesions subject to this type of repair include cyclobutane pyrimidine dimers that are normally repaired by nucleotide excision repair (NER) and thymine glycols (TGs) that are removed primarily by base excision repair (BER). While the mechanism underlying TCR is not completely clear, it is known that its facilitation requires proteins used by other repair pathways like NER. It is also believed that the signal for TCR is the stalled RNA polymerase that results when DNA damage prevents its translocation during transcription elongation. While there is a clear role for some NER proteins in TCR, the involvement of BER proteins is less clear. To explore this further, we studied the removal of 7-methylguanine (7MeG) and 3-methyladenine (3MeA) from the dihydrofolate reductase (dhfr) gene of murine cell lines that vary in their repair phenotypes. 7MeG and 3MeA constitute the two principal N-methylpurines formed in DNA following exposure to methylating agents. In mammalian cells, alkyladenine DNA alkyladenine glycosylase (Aag) is the major enzyme required for the repair of these lesions via BER, and their removal from the total genome is quite rapid. There is no observable TCR of these lesions in specific genes in DNA repair proficient cells; however, it is possible that the rapid repair of these adducts by BER masks any TCR. The repair of 3MeA and 7MeG was examined in cells lacking Aag, NER, or both Aag and NER to determine if rapid overall repair masks TCR. The results show that both 3MeA and 7MeG are removed without strand bias from the dhfr gene of BER deficient (Aag deficient) and NER deficient murine cell lines. Furthermore, repair of 3MeA in this region is highly dependent on Aag, but repair of 7MeG is equally efficient in the repair proficient, BER deficient, and NER deficient cell lines. Strikingly, in the absence of both BER and NER, neither 7MeG nor 3MeA is repaired. These results demonstrate that NER, but not TCR, contributes to the repair of 7MeG, and to a lesser extent 3MeA.

Transcription-coupled repair of DNA adducts is an essential factor that must be considered when one is elucidating biological endpoints resulting from exposure to genotoxic agents. Alkylating agents comprise one group of chemical compounds which modify DNA by reacting with oxygen and nitrogen atoms in the bases of the double helix. To discern the role of transcription-coupled DNA repair of N-ethylpurines present in discrete genetic domains, Chinese hamster ovary cells were exposed to N-ethyl-N-nitrosourea, and the clearance of the damage from the dihydrofolate reductase gene was investigated. The results indicate that N-ethylpurines were removed from the dihydrofolate reductase gene of nucleotide excision repair-proficient Chinese hamster ovary cells; furthermore, when repair rates in the individual strands were determined, a statistically significant bias in the removal of ethyl-induced, alkali-labile sites was observed, with clearance occurring 30% faster from the transcribed strand than from its nontranscribed counterpart at early times after exposure. In contrast, removal of N-ethylpurines was observed in the dihydrofolate reductase locus in cells that lacked nucleotide excision repair, but both strands were repaired at the same rate, indicating that transcription-coupled clearance of these lesions requires the presence of active nucleotide excision repair.

The mutagenic and carcinogenic consequences of unrepaired DNA damage depend upon its precise location with respect to the relevant genomic sites. Therefore, it is important to learn the fine structure of DNA damage, in particular, proto-oncogenes, tumor-suppressor genes, and other DNA sequences implicated in tumorigenesis. Both the introduction and the repair of many types of DNA lesions are heterogeneous with respect to chromatin structure and/or gene activity. For example, cyclobutane pyrimidine dimers are removed more efficiently from the transcribed than the nontranscribed strand of the dhfr gene in Chinese hamster ovary cells. In contrast, preferential strand repair of alkali-labile sites is not found at this locus. In mouse 3T3 cells, dimers are more efficiently removed from an expressed proto-oncogene than from a silent one. Persistent damage in nontranscribed domains may account for genomic instability in those regions, particularly during cell proliferation as lesions are encountered by replication forks. The preferential repair of certain lesions in the transcribed strands of active genes results in a bias toward mutagenesis owing to persistent lesions in the nontranscribed strands. Risk assessment in environmental genetic toxicology requires assays that determine effective levels of DNA damage of producing malignancy. The existence of nonrandom repair in the mammalian genome casts doubt on the reliability of overall indicators of carcinogen-DNA binding and lesion repair for such determinations. Tissue-specific and cell-specific differences in the coordinate regulation of gene expression and DNA repair may account for corresponding differences in the carcinogenic response to particular environmental agents.

Alkylating agent damage was quantified in human T-lymphocytes by calculating gene-specific lesion frequencies and repair rates. At 3 time points after exposure to methyl methanesulfonate (0, 6, and 24 h), T-lymphocyte DNA was extracted, digested with HindIII, and divided into 2 aliquots. Apurinic sites were formed in the DNA fragments of both aliquots by heat-induced liberation of the N-methylpurines. The methoxyamine-treated aliquot provided gene fragments which were refractory to alkaline hydrolysis (full-length fragments), while the fragments in the untreated aliquot were cleaved at apurinic sites by hydroxide. After Southern blotting, lesion frequencies were calculated by comparing the band intensity of the full-length fragment to its unprotected counterpart. The restriction fragments analyzed were from the constitutively active dihydrofolate reductase (dhfr) plus hypoxanthine phosphoribosyltransferase (hprt) genes and from the transcriptionally inactive Duchenne muscular dystrophy gene (dmd). In decreasing order, the fragments containing the most lesions per kb of DNA were: hprt greater than dhfr greater than dmd. T-Lymphocytes from 2 females had 30% more heat-labile N-methylpurines in the active X-linked hprt gene than in the inactive X-linked dmd gene. The lesion frequency found in the male's lone hprt allele was the highest observed. These lesion frequency differences are discussed in terms of chromatin structure. After 6 and 24 h, no significant repair rate differences were observed among the 3 genes.

The removal of N-methylpurines from the DHFR gene and an unexpressed adjacent locus located downstream occurs at similar rates and to a similar extent in dimethyl sulfate treated Chinese hamster ovary B11 cells. Furthermore, no significant differences in repair rates are observed between the strands of the active gene. These data primarily reflect the removal of the most abundant lesion produced by dimethyl sulfate, 7-methylguanine, and are in contrast to the results obtained for the removal of ultraviolet-induced cyclobutane pyrimidine dimers from the same region of the genome. Pyrimidine dimers are cleared preferentially from the transcribed strand of the DHFR gene and are removed poorly from the non-transcribed complementary strand and unexpressed adjacent regions. The results suggest that DNA lesions such as dimers that block transcription are removed preferentially from active genes, whereas lesions that do not interfere with nucleic acid synthesis (i.e. 7-methylguanine) are removed at similar rates from expressed and silent loci.

The activity of the DNA-repair protein O6-alkylguanine-DNA-alkyltransferase was found to be strongly inhibited by a number of metal ions. Cd2+ was the most active followed by Cu2+, Hg2+, Zn2+ and Ag2. This inhibition is likely to result from the interaction of the metals with the cysteine-acceptor residue on the protein since the inhibition was reduced by increasing the concentration of dithiothreitol in the assay buffer. These results raise the possibility that exposure to Cd2+ could increase the mutagenicity and carcinogenicity of alkylating agents by retarding the rate of repair of alkylated DNA. However, other metals or metallic compounds which are known to be carcinogenic (such as compounds containing arsenic, lead, nickel or chromium) did not interfere with DNA repair by this protein.

We have developed a quantitative method for examining the removal of N-methylpurines from specific genes to investigate their possible differential repair throughout the genome. Chinese hamster ovary cells were exposed to dimethyl sulfate, and the isolated DNA was treated with an appropriate restriction endonuclease. The DNA was heated to convert remaining N-methylpurines to apurinic sites to render them alkaline-labile. Duplicate samples heated in the presence of methoxyamine to protect the apurinic sites from alkaline hydrolysis provided controls to assess total DNA. After alkaline hydrolysis, agarose gel electrophoresis, Southern transfer, and probing for the fragment of interest, the ratios of band intensities of the test DNA sample to its methoxyamine-treated control counterpart were calculated to yield the percentage of fragments containing no alkaline-labile sites. The frequency of N-methylpurines was measured at different times after dimethyl sulfate treatment to study repair. We found no differences between the rates of repair of N-methylpurines in the active dihydrofolate reductase gene and a nontranscribed region located downstream from it in treated cells. Also, similar rates of repair were observed in the transcribed and nontranscribed strands of the gene, in contrast to previous results for the removal of cyclobutane pyrimidine dimers. Thus, there does not appear to be a coupling of N-methylpurine repair to transcription in Chinese hamster ovary cells. However, the repair in the dihydrofolate reductase domain appears to be somewhat more efficient than that in the genome overall. Our method permits the quantifying at the defined gene level of abasic sites or of any DNA adduct that can be converted to them.