Reference #: PFE-1018-533234
Submit Date: 04/11/2002 08:49:09-0500

Mutations induced by UV and sunlight

ABSTRACT:
A hallmark of UV mutagenesis is the high frequency of transition mutations at dipyrimidine sequences containing cytosine. In human skin cancers, more than 30% of all mutations in the p53 gene are transitions at dipyrimidines within the sequence context CpG, i.e. 5'-TCG and 5'-CCG, found at several mutational hotspots. Since CpGs are methylated along the p53 gene, these mutations may be derived from solar UV-induced pyrimidine dimers forming at sequences that contain 5-methylcytosine. Previously, it had been shown that CPDs form preferentially at dipyrimidines containing 5-methylcytosine when UVB (Drouin and Therrien, 1997) or sunlight (Tommasi et al., 1997) was used for irradiation. In order to define the contribution of 5-methylcytosine to sunlight-induced mutations, the CpG-methylated lacI and cII transgenes in mouse fibroblasts were used as mutational targets. After UVC irradiation, 6-9% of the base substitutions were at dipyrimidines containing 5-methylcytosine and only few mutations were transitions within this sequence context. However, 24-32% of the solar light-induced mutations were at dipyrimidines that contain 5-methylcytosine and most of them were transitions. Sunlight-induced mutational hotspots at methylated CpGs correlated with sequences that form high levels of CPDs after irradiation with sunlight but not with UVC. The data make a strong case that CPDs forming preferentially at dipyrimidines with 5-methylcytosine are responsible for a considerable fraction of the mutations induced by sunlight in mammalian cells. Using photoproduct-specific photolyases transfected into cells containing mutational reporter genes, we demonstrated that CPDs (rather than 6-4-products or other lesions) are indeed responsible for the vast majority of UVB-induced mutations in mammalian cells. Current work is aimed at studying the mechanisms of mutagenesis by CPDs containing cytosine and 5-methylcytosine.

Keywords: sunlight, mutation, cyclobutane dimer, (6-4) photoproduct

Reference #: SME-1016-405641
Submit Date: 03/17/2002 15:56:47-0500

DNA Repair in RNA Polymerase I Transcribed Genes

ABSTRACT:
UV-induced cyclobutane pyrimidine dimers (CPDs) in DNA are substrates for two sub-pathways of nucleotide excision repair (NER): transcription-coupled repair (TCR) and global genome repair. TCR is efficient at removing CPDs from the transcribed strand (TS) of actively transcribing RNA polymerase II genes. Conversely, in mammalian cells, TCR has not been observed in genes transcribed by RNA polymerases I (Pol I) and III, and NER of CPDs in mammalian ribosomal RNA genes (rDNA) is very inefficient. On the other hand, CPDs are rapidly repaired in the ~150 tandem repeated rDNA in yeast. Moreover, strand-specific repair has been found in rad7 and rad16 mutants, suggesting that TCR may exist in yeast rDNA. However, prior studies analyzed a mixed population of transcribed and non-transcribed rRNA genes, and a definitive demonstration of whether TCR occurs in Pol I genes was not possible. To determine if TCR is present in Pol I transcribed genes, NER of UV induced CPDs was measured in the individual strands of transcriptionally active and inactive rDNA of yeast. Restriction enzyme digestion and selective psoralen cross-linking was used to distinguish between active- and inactive-rDNA chromatin. The data clearly show that TCR occurs in Pol I transcribed genes in yeast. Moreover, the NTS of active rDNA is repaired faster than either strand of inactive rDNA, implying that NER has preferential access to the active rDNA chromatin. Finally, psoralen cross-linking of active rDNA varies during NER, suggesting there is a change in rDNA chromatin structure during (or soon after) CPD removal. Active rDNA copies are initially folded into a very compact chromatin during, or soon after, repair of CPDs. After longer times, however, there is a gradual unfolding of the compact rDNA chromatin back to a more open conformation. Transcription of rDNA is sharply reduced following UV irradiation and remains at reduced levels during DNA repair in hydroxyurea-treated yeast cells. Therefore, changes in rRNA transcription in do not correlate with the changes in chromatin structure following UV damage and repair. These results reveal that (1) TCR occurs in Pol I transcribed rRNA genes in yeast, (2) both strands of active rDNA are repaired faster than the inactive rDNA, and (3) chromatin remodeling may be due to NER in the ribosomal gene locus.

Keywords: chromatin, excision repair

Reference #: TAY-1017-721043
Submit Date: 04/01/2002 22:04:08-0500

Mechanistic study of photoproduct bypass by polymerase eta.

ABSTRACT:
We are interested in understanding the structural and mechanistic basis for the origin of mutations induced by UV photoproducts. In particular we are interested in understanding why a DNA damage bypass polymerase like E. coli pol V, can easily bypass cis-syn dimers, whereas a replicative polymerase like T7 DNA polymerase cannot. Furthermore we are interested in understanding why pol V inserts primarily A opposite the 3'-T of a cis-syn dimer of TT, and G opposite the 3'-T of a (6-4) product of TT, whereas T7 DNA polymerase, inserts primarily A opposite the 3'-T of both the cis-syn and (6-4) products of TT. Examination of the crystal structure of a complex between T7 DNA polymerase, a template-primer and a ddNTP reveals that the base templating the addition of a nucleotide is flanked the 3'-side by a base, and on the 5'-side by the O-helix of the polymerase. As a result, the nucleotide to the 5'-side of the templating base is excluded from the active site, thereby resulting in a bent template. Based on this structure, we proposed that a dipyrimidine photoproduct cannot be accommodated by the active during insertion opposite the 3'-T, and insertion of A takes place because of the inherent preference for inserting A opposite an empty template site, typified by an abasic site (Smith et al., J Biol Chem 1998 273, 21933. Further support for this "transient abasic site-like" bypass mechanism comes from nucleotide insertion assays with pyrene nucleotide which is preferentially inserted opposite abasic sites by T7 polymerase (Sun et al., Biochemistry 2000 39, 14603. In this talk we will describe a variety of mechanistic studies on the DNA damage bypass polymerase, yeast pol eta, a member of the Y family polymerases to which E. coli pol V belongs. This polymerase can bypass cis-syn TT dimers easily and in an error-free manner. Defective forms of the human version of this polymerase have been implicated in the genetic disease Xeroderma pigmentosum variant which leads to a higher incidence of skin cancer. We find that unlike the replicative T7 DNA polymerase, yeast pol eta appears to be able to accommodate the cis-syn dimer of TT in the active site during insertion opposite the 3'-T. The implications of the bypass mechanism of this polymerase for the orgin of UV-induced mutations in humans will also be discussed. (This work was funded by NIH CA40463).

Keywords: photoproduct, polymerase, mutagenesis, mechanism

Reference #: 080118
Submit Date:

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Reference #: DAV-1017-060756
Submit Date: 03/25/2002 06:03:25-0500

Profiles and products of photosensitized DNA damage.

ABSTRACT:
We have utilised the complementary approaches of gel sequencing experiments with synthetic oligonucleotides, and product analysis with nucleoside model compounds, to investigate the action of heterocyclic and ketonic photosensitizers towards DNA. Similar profiles of piperidine-labile base damage are produced in end-labeled DNA molecules photosensitized with derivatives of phenylbenzimidazole, certain aryl propionate drugs, and nalidixic acid. In every case, the cleavage sites map almost exclusively to guanine (G) bases but the reactivity patterns for single-stranded and duplex DNA are quite different. In the former case, where there is uniform attack at all G bases, the profile is compatible with a Type II process mediated by singlet oxygen. With duplex DNA, however, damage is strongly localised to G bases that are 5'- to an adjacent purine as predicted for a Type I mechanism involving electron transfer from DNA to the sensitizer. By contrast, in behavior suggesting free radical attack, photosensitization with the fluoroquinolone lomefloxacin induces alkali-labile lesions essentially at random in both single- and double-stranded DNA. To corroborate inferences drawn from DNA sequencing profiles, the capacity of individual compounds to photooxidize G residues by a Type I and/or Type II mechanism has been tested by irradiating them with 2'-deoxyguanosine (dG) then using HPLC to assay respectively for formation of the deoxyribonucleosides of spiroiminodihydantoin (Sp) and diaminoimidazolone (Iz). As some cyclic peroxides have proved useful for mimicking specific photooxidative mechanisms, we have extended earlier studies of dimethyldioxirane (DMD) in this context. Treatment of dG with DMD initiates a new oxidative pathway leading ultimately to 2-(2,3,4-trihydroxybutyl)-4-amidinocarbamoyl-5-hydroxyimidazole. It is particularly noteworthy that analogous incorporation of a trihydroxybutyl substituent into the guanine nucleus occurs when dG undergoes anaerobic photosensitization with acetone. The mechanistic basis for this unusual parallel between the two reactions, and its relevance at the polynucleotide level, is being investigated.

Keywords: DNA sequencing, guanine, oxidative damage, photosensitizers

Reference #: CAD-1017-330595
Submit Date: 03/28/2002 09:07:47-0500

UV-B and UV-A induced formation of photoproducts within cellular DNA: direct and photosensitized effects

ABSTRACT:
UV-B AND UV-A INDUCED FORMATION OF PHOTOPRODUCTS WITHIN CELLULAR DNA: DIRECT AND PHOTOSENSITIZED EFFECTS. J. Cadet, T. Douki, J.-P. Pouget and J;-L. Ravanat, SCIB/LAN & UMR 5046, CEA/Grenoble, 38054-Grenoble, France It is well established that UV-B and UV-A act mostly on cellular DNA via direct and photosensitized reactions respectively. Precise assessment of the photoproducts of the latter reactions has been hampered for years by lack of accurate and quantitative methods of measurement. This particularly applies to the individual determination of dimeric photoproducts including the cis-syn cyclobutadipyrimidines, the pyrimidine (6-4) pyrimidone photoadducts and their related valence Dewar isomers. Interestingly the individual measurement of the twelve possible dimeric photoproducts as dinucleoside monophosphates at the four main dipyrimidine sites is now possible. This is achieved using a specific and sensitive assay that couples high performance liquid chromatography to tandem mass spectrometry (HPLC-MS/MS). Strikingly it was found that UV-B irradiation of human monocyte cells gives rise predominantly to cis-syn cyclobutadithymine and the thymine-cytosine (6-4) adduct. On the other hand the dimeric photoproducts at dicytosine sites are minimally generated although characteristic tandem mutations of UV-B irradiation are observed at the latter CC sequences. Further, cytosine photohydrate and Dewar valence isomers of the (6-4) photoproducts are at the best minor UV-B photoproducts. Relevant information on UVA- sensitized oxidative damage to cellular DNA was gained from measurements achieved with chromatographic methods and the modified comet assay. Thus it was shown that 8-oxo-7,8-dihydro-2'-deoxyguanosine is the predominant oxidation product as mostly the result of singlet oxygen reaction. In addition, oxidized pyrimidine bases and DNA strand breaks whose formation involves OH radical are produced in lower yields. Work is in progress to assess the UVA-induced formation of other markers of oxidative stress (DNA protein crosslinks and DNA adducts with reactive aldehydes that arise from the breakdown of lipid peroxidation).

Keywords: nucleic acid photodamage, dimeric pyrimidine lesions, oxidized base damage, singlet oxygen