David A. Scicchitano

Base excision repair and nucleotide excision repair contribute to the removal of N-methylpurines from active genes

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.

Comparison of repair of methylated pyrimidines in poly(dT) by extracts from rat liver and Escherichia coli

Partially purified preparations of O6-alkylguanine-DNA alkyltransferase from rat liver and E. coli were tested for their ability to repair O4-methylthymine in a methylated poly(dT) X poly(dA) substrate. The bacterial preparation readily carried out this reaction, but no loss of O4-methylthymine was obtained with the rat liver protein. These results indicate a significant difference in specificity between the mammalian and bacterial proteins which could have important consequences for carcinogenesis and mutagenesis by alkylating agents in mammalian cells.

Benzo[ a ]pyrene diol epoxide stimulates an inflammatory response in normal human lung fibroblasts through a p53 and JNK mediated pathway

Cellular responses to carcinogens are typically studied in transformed cell lines, which do not reflect the physiological status of normal tissues. To address this question, we have characterized the transcriptional program and cellular responses of human lung WI-38 fibroblasts upon exposure to the ultimate carcinogen benzo[a]pyrene diol epoxide (BPDE). In contrast to observations in cell lines, we find that BPDE treatment induces a strong inflammatory response in these normal fibroblasts. Whole-genome microarrays show induction of numerous inflammatory factors, including genes that encode interleukins (ILs), growth factors and enzymes related to prostaglandin synthesis and signaling. Real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) revealed a time- and dose-dependent-induced expression and production of cyclooxygenase 2, prostglandin E2 and IL1B, IL6 and IL8. In parallel, cell cycle progression and DNA repair processes were repressed, but DNA damage signaling was increased via p53-Ser15 phosphorylation and induced expression levels of GADD45A, CDKN1A, BTG2 and SESN1. Network analysis suggested that activator protein 1 transcription factors may link the cell cycle response and DNA damage signaling with the inflammatory stress-response in these cells. We confirmed this hypothesis by showing that p53-dependent signaling through c-jun N-terminal kinase (JNK) led to increased cJun-Ser63 phosphorylation and that inhibition of JNK-mediated cJun activation using p53- or JNK-specific inhibitors significantly reduced IL gene expression and subsequent production of IL8. This is the first demonstration that a strong inflammatory response is triggered in normal fibroblasts by BPDE and that this occurs through coordinated regulation with other cellular processes.

Inhibition of O6-alkylguanine-DNA-alkyltransferase by metals

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.

O6-Methylguanine induces altered proteins at the level of transcription in human cells

O6-Methylguanine (O6-meG), which is produced in DNA following exposure to methylating agents, instructs human RNA polymerase II to mis-insert bases opposite the lesion during transcription. In this study, we examined the effect of O6-meG on transcription in human cells and investigated the subsequent effects on protein function following translation of the resulting mRNA. In HEK293 cells, O6-meG induced incorporation of uridine or cytidine in nascent RNA opposite the adduct. In cells containing active O6-alkylguanine-DNA alkyltransferase (AGT), which repairs O6-meG, 3% misincorporation of uridine was observed opposite the lesion. In cells where AGT function was compromised by addition of the AGT inhibitor O6-benzylguanine, ∼58% of the transcripts contained a uridine misincorporation opposite the lesion. Furthermore, the altered mRNA induced changes to protein function as demonstrated through recovery of functional red fluorescent protein (RFP) from DNA coding for a non-fluorescent variant of RFP. These data show that O6-meG is highly mutagenic at the level of transcription in human cells, leading to an altered protein load, especially when AGT is inhibited.

Functional nucleotide excision repair is required for the preferential removal of N-ethylpurines from the transcribed strand of the dihydrofolate reductase gene of Chinese hamster ovary cells.

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.

Transcription elongation past O6-methylguanine by human RNA polymerase II and bacteriophage T7 RNA polymerase

O6-Methylguanine (O6-meG) is a major mutagenic, carcinogenic and cytotoxic DNA adduct produced by various endogenous and exogenous methylating agents. We report the results of transcription past a site-specifically modified O6-meG DNA template by bacteriophage T7 RNA polymerase and human RNA polymerase II. These data show that O6-meG partially blocks T7 RNA polymerase and human RNA polymerase II elongation. In both cases, the sequences of the truncated transcripts indicate that both polymerases stop precisely at the damaged site without nucleotide incorporation opposite the lesion, while extensive misincorporation of uracil is observed in the full-length RNA. For both polymerases, computer models suggest that bypass occurs only when O6-meG adopts an anti conformation around its glycosidic bond, with the methyl group in the proximal orientation; in contrast, blockage requires the methyl group to adopt a distal conformation. Furthermore, the selection of cytosine and uracil partners opposite O6-meG is rationalized with modeled hydrogen-bonding patterns that agree with experimentally observed O6-meG:C and O6-meG:U pairing schemes. Thus, in vitro, O6-meG contributes substantially to transcriptional mutagenesis. In addition, the partial blockage of RNA polymerase II suggests that transcription-coupled DNA repair could play an auxiliary role in the clearance of this lesion.

Two expressed human genes sustain slightly more DNA damage after alkylating agent treatment than an inactive gene

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 males 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.

Lack of sequence-specific removal of N-methylpurines from cellular DNA

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.

Transcription of DNA containing the 5-guanidino-4-nitroimidazole lesion by human RNA polymerase II and bacteriophage T7 RNA polymerase

Damage in transcribed DNA presents a challenge to the cell because it can partially or completely block the progression of an RNA polymerase, interfering with transcription and compromising gene expression. While blockage of RNA polymerase progression is thought to trigger the recruitment of transcription-coupled DNA repair (TCR), bypass of the lesion can also occur, either error-prone or error-free. Error-prone transcription is often referred to as transcriptional mutagenesis (TM). Elucidating why some lesions pose blocks to transcription elongation while others do not remains a challenging problem. As part of an effort to understand this, we studied transcription past a 5-guanidino-4-nitroimidazole (NI) lesion, using two structurally different RNA polymerases, human RNA polymerase II (hRNAPII) and bacteriophage T7 RNA polymerase (T7RNAP). The NI damage results from the oxidation of guanine in DNA by peroxynitrite, a well known, biologically important oxidant. It is of structural interest because it is a ring-opened and conformationally flexible guanine lesion. Our results show that NI acts as a partial block to T7RNAP while posing a major block to hRNAPII, which has a more constrained active site than T7RNAP. Lesion bypass by T7RNAP induces base misincorporations and deletions opposite the lesion (C>A>-1 deletion >G >>> U), but hRNAPII exhibits error-free transcription although lesion bypass is a rare event. We employed molecular modeling methods to explain the observed blockage or bypass accompanied by nucleotide incorporation opposite the lesion. The results of the modeling studies indicate that NIs multiple hydrogen-bonding capabilities and torsional flexibility are important determinants of its effect on transcription in both enzymes. These influence the kinetics of lesion bypass and may well play a role in TM and TCR in cells.