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G P Pfeifer

Publications and source records attributed to G P Pfeifer.

At least 19 recordsLinked to original sources
PubMed1996

Inhibition of transcription factor binding by ultraviolet-induced pyrimidine dimers.

The formation of DNA photoproducts by ultraviolet (UV) light is responsible for the induction of mutations and the development of skin cancer. Cis-syn cyclobutane pyrimidine dimers (pyrimidine dimers) are the most frequent lesions produced in DNA by UV irradiation. Besides being mutagenic, pyrimidine dimers may interfere with other important DNA-dependent processes. To analyze the effects of pyrimidine dimers on the ability of DNA sequences to be recognized by trans-acting factors, we have incorporated site-specific T-T dimers into oligonucleotides containing the recognition sequences of the sequence-specific transcription factors E2F, NF-Y, AP-1, NF kappa B, and p53. In each case, presence of the photodimer strongly inhibited binding of the respective transcription factor complex. Reduction of binding varied between 11- and 60-fold. The results indicate that the most common UV-induced DNA lesion can interfere severely with binding of several important cell cycle regulatory and DNA damage responsive transcription factors. We suggest that inhibition of transcription factor binding may be a major biological effect of UV radiation since promoter regions are known to be repaired inefficiently and since UV damage can deregulate the function of a large number of different factors.

Binding Sites↗
PubMed1996

Preferential formation of benzo[a]pyrene adducts at lung cancer mutational hotspots in P53.

Cigarette smoke carcinogens such as benzo[a]pyrene are implicated in the development of lung cancer. The distribution of benzo[a]pyrene diol epoxide (BPDE) adducts along exons of the P53 gene in BPDE-treated HeLa cells and bronchial epithelial cells was mapped at nucleotide resolution. Strong and selective adduct formation occurred at guanine positions in codons 157, 248, and 273. These same positions are the major mutational hotspots in human lung cancers. Thus, targeted adduct formation rather than phenotypic selection appears to shape the P53 mutational spectrum in lung cancer. These results provide a direct etiological link between a defined chemical carcinogen and human cancer.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1996

DNA repair domains within a human gene: selective repair of sequences near the transcription initiation site.

We describe a new form of DNA repair heterogeneity along the genome. The repair rate of UV-induced cyclobutane pyrimidine dimers (CPDs) was measured at single nucleotide resolution along the promoter and transcribed sequences of the human JUN gene in UV-irradiated diploid fibroblasts. The promoter of this gene contains an array of sequence-specific transcription factors located between nucleotides -200 and -50 relative to the major transcription start site. These sequences are repaired slowly; at many sites >50% of the CPDs are left unrepaired after 24 h. However, repair rates are 10-fold faster near the transcription initiation site. This very fast repair is seen on both DNA strands between nucleotides -40 and +100 where at most positions >90% of the dimers are repaired within 4 h. There is a general gradient of repair efficiency of the transcribed DNA strand with faster repair within the 5'-end and diminished repair towards the 3'-end of the gene. The fast repair rates seen near the transcription initiation site may be explained by increased local concentrations of DNA repair factors that are associated with general transcription factors (e.g. TFIIH) functioning in transcription initiation. This domain-specific DNA repair may aid in maintaining transcription initiation of essential genes after DNA damage.

Base Sequence↗
PubMed1996

UV damage and repair mechanisms in mammalian cells.

The formation of DNA photoproducts by ultraviolet (UV) light is responsible for induction of mutations and development of skin cancer. To understand UV mutagenesis, it is important to know the mechanisms of formation and repair of these lesions. Cyclobutane pyrimidine dimers and (6-4)photoproducts are the two major classes of UV-induced DNA lesions. Their distribution along DNA sequences in vivo is strongly influenced by nucleosomes and other DNA binding proteins. Repair of UV photoproducts is dependent on the transcriptional status of the sequences to be repaired and on the chromatin environment. Sensitive techniques are now available to study repair of UV damage at the level of nucleotide resolution in mammalian cells. With the aid of in vitro systems, the entire nucleotide excision repair process has been reconstituted from purified protein components with naked DNA as a substrate. Future work will focus on the development of in vitro assays for transcription-coupled repair and repair in chromatin.

Animals↗
PubMed1996

Genomic sequencing by ligation-mediated PCR.

Genomic sequencing permits studies of in vivo DNA methylation and protein-DNA interactions, but its use has been limited due to the complexity of the mammalian genome. Ligation-mediated PCR (LMPCR) is a sensitive genomic sequencing procedure that generates high quality, reproducible sequence ladders starting with only 1 microgram of uncloned mammalian DNA per reaction. This genomic sequencing procedure can be adapted for various methylation, in vivo footprinting and DNA adduct mapping procedures. We provide a detailed protocol for genomic sequencing by LMPCR and discuss the principles and applications of the method.

DNA Adducts↗
PubMed1995

UV light as a footprinting agent: modulation of UV-induced DNA damage by transcription factors bound at the promoters of three human genes.

Protein-DNA interactions in mammalian cells can be analyzed at the nucleotide level of resolution by genomic sequencing techniques. The most sensitive genomic sequencing method uses the ligation-mediated polymerase chain reaction (LMPCR) for signal amplification to detect the positions of DNA modifications or strand breaks. Various probing methods are compatible with LMPCR, but dimethyl sulfate footprinting has most commonly been used. Here, we have examined the suitability of ultraviolet (UV) light as an in vivo footprinting agent to detect a wide variety of protein-DNA contacts. The distribution of the two major types of UV-induced DNA photoproducts (cyclobutane pyrimidine dimers and (6-4) photo-products) has been examined along the promoter sequences of three human genes. A comparison of UV-irradiated naked DNA and UV-irradiated cells reveals differences in the UV damage spectrum for both types of photoproducts. These differences can be either decreases or dramatic increases of photoproduct frequency. At the promoter of the c-jun gene, these differences ("photofootprints") co-localize with binding sites for two AP-1-like transcription factors, a CCAAT box binding protein, an SP-1 sequence, an NF-jun sequence, a related to serum response factor (RSRF) binding site and a sequence bound by an unknown factor. In the promoter of the gene coding for proliferating cell nuclear antigen (PCNA), photofootprints were seen at two SP-1 like sequences and two CCAAT boxes. The c-fos promoter is characterized by photofootprints at the serum response element (SRE), at the adjacent binding site for ternary complex factor (TCF), at an AP-1 site and at a binding site for a growth factor inducible protein (SIF). Photofootprints may be signatures of specific transcription factors or families of related factors since we noticed that the photofootprints seen at several common factor binding sites were similar or identical when the same site was analyzed in different genes. Photofootprints were not seen at sequences distant from transcription factor binding sites. A comparison of our UV photofootprinting data with data from experiments using other probing strategies shows that UV light has the potential to reveal all protein-DNA interactions provided there is a dipyrimidine sequence on either DNA strand within a factor binding site. The simplicity of using this probing agent together with its specificity for detecting a large variety of different factors should make UV light a generally useful tool for in vivo footprinting studies.

Base Sequence↗
PubMed1995

Complete and tissue-independent methylation of CpG sites in the p53 gene: implications for mutations in human cancers.

CpG dinucleotides are the target of about one third of transition mutations found in human genetic diseases and tumors. Methylation at these sites is thought to be the cause of these genetic changes through spontaneous deamination of 5-methylcytosine. In order to define the contribution of 5-methylcytosine to the spectrum of p53 mutations in human cancers, we have determined the complete DNA methylation pattern along exons 5-8 of the human p53 gene by ligation-mediated polymerase chain reaction genomic sequencing. The study was conducted with nine different types of normal human tissue and cell lines, including skin fibroblasts, keratinocytes, lung epithelial cells, mammary epithelial cells and colonic mucosa cells. We found that the p53 sequences along exons 5-8 are completely methylated at every CpG site, including 46 different sites on both DNA strands. This methylation pattern is tissue-independent suggesting that tissue-specific methylation does not contribute to the differential mutation patterns seen in tumors. The occurrence of mutational hotspots at specific CpG sites is not related to selective methylation of only a subset of CpGs but may rather depend on a selection bias for particular amino acid changes. Our results are not inconsistent with theories that mutations in tumors with high CpG mutation rates, like colon cancer, are caused by spontaneous deamination of 5-methylcytosine and mutations in tumors with a lack of CpG involvement reflect superimposed fingerprints from exogenous carcinogens. However, given the lack of tissue specificity of methylation, alternative explanations (eg targeting of methylated CpG sites by tissue-selective carcinogens) should be considered to explain the high percentage of CpG mutations in some tumor types.

Base Sequence↗
PubMed1995

In vivo protein-DNA interactions at the c-jun promoter in quiescent and serum-stimulated fibroblasts.

c-Jun is an important component in the regulation of cell proliferation. As a member of the early response gene family, c-jun is induced within minutes in the presence of mitogenic agents such as serum growth factors. Using in vivo footprinting, we have analyzed protein-DNA interactions at the c-jun promoter in human fibroblasts subjected to growth arrest and serum stimulation. We located seven footprints upstream of the transcription initiation site. Protein-DNA interactions were detected at two AP-1-like sequences, A CCAAT box, an SP-1 sequence, an NF-jun sequence, a putative RSRF (related to serum response factor) binding site, and a sequence bound by an unknown factor. All of these binding sites were occupied in serum-starved cells, and no additional protein-DNA interactions were detected upon serum stimulation. Evidence from this study supports a model in which expression of the c-jun gene is mediated by phosphorylation events taking place on the transactivation domains of promoter-bound transcriptional activators.

Base Sequence↗
PubMed1995

In vivo structure of the human cdc2 promoter: release of a p130-E2F-4 complex from sequences immediately upstream of the transcription initiation site coincides with induction of cdc2 expression.

In quiescent cells, cdc2 mRNA is almost undetectable. Stimulation of cells to reenter the cell cycle results in induction of cdc2 expression, beginning at the G1-to-S transition and reaching maximum levels during late S and G2 phases. To investigate cdc2 transcriptional regulation throughout cell cycle progression, we monitored protein-DNA interactions by in vivo footprinting along 800 bp of the human cdc2 promoter in quiescent fibroblasts and at different time points following serum stimulation. We found 11 in vivo protein-binding sites, but no protein binding was observed at a high-affinity E2F site that had previously been implicated in cdc2 regulation. Nine of the identified in vivo binding sites (among them were two inverted CCAAT boxes, two Sp1 sites, and one ets-2 site) bind transcription factors constitutively throughout the cell cycle. However, at two elements located at positions -60 and -20 relative to the transcription start site, the binding pattern changes significantly as the cells are entering S phase. A G0- and G1-specific protein complex disappears at the -20 element at the beginning of S phase. This sequence deviates at one base position from known E2F consensus binding sites. We found that the major E2F activity in human fibroblasts contains E2F-4 and p130. The -20 element of the cdc2 gene specifically interacts with a subset of E2F-4-p130 complexes present in G0 cells but does not interact with S-phase-specific E2F complexes. Transient-transfection experiments with wild-type and mutant cdc2 promoter constructs indicate that the -20 element is involved in suppressing cdc2 activity in quiescent cells. We suggest that the presence of the p130-E2F-4 complex in G0/G1 blocks access of components of the basal transcription machinery or prevents transaction by the constitutively bound upstream activator proteins.

Base Sequence↗
PubMed1994

Mapping of DNA alkylation sites induced by adozelesin and bizelesin in human cells by ligation-mediated polymerase chain reaction.

In this study, we have mapped the intracellular alkylation sites of adozelesin and bizelesin, two potent analogs of CC-1065, in individual genes at the single-nucleotide level. Human colon carcinoma cells were treated with adozelesin and bizelesin, and the position of adducts were mapped within the PGK-1 and p53 genes by means of ligation-mediated polymerase chain reaction. The monofunctional alkylating agent adozelesin was found to alkylate genomic DNA predominantly within 5'-(A/T)(A/T)A* sequences. Additional sites of alkylation were observed within 5'-(A/T)(G/C)(A/T)A* sequences; however, these were considered to represent sites of medium to low preference. Bizelesin, a bifunctional analog capable of both DNA monofunctional alkylation and DNA interstrand cross-link formation, was also found to alkylate 5'-(A/T)(A/T)A* sequences. Putative bizelesin DNA interstrand cross-link sites indicated that AT-rich sequences are preferred in the intervening sequence between the two cross-linked adenines. Both six- and seven-nucleotide regions were identified as putative sites of DNA interstrand cross-link formation with 5'-TTTTTTA*, 5'-TTTATCA* and 5'-GTACTAA* sequences being preferred. Non-adenine bases are not observed as potential intracellular sites of either DNA interstrand cross-linking formation or monofunctional alkylation. Thus, the patterns of alkylation induced by adozelesin and bizelesin in genomic DNA are similar but not identical to that observed in purified cell-free DNA.

Alkylating Agents↗
PubMed1994

Mapping of DNA alkylation sites induced by aziridinylbenzoquinones in human cells by ligation-mediated polymerase chain reaction.

Diaziridinylbenzoquinones such as 3,6-diaziridinyl-1,4-benzoquinone (DZQ) and its 2,5-methyl analog (MeDZQ) require bioreductive activation in order to elicit their cytotoxic activities. In this study, we have mapped the intracellular alkylation sites induced by DZQ and MeDZQ in a single copy gene at the nucleotide level using ligation-mediated polymerase chain reaction. We have performed this analysis in two human colon carcinoma cells, one proficient (HT-29) and one deficient (BE) in DT-diaphorase (DTD) activity. In the DTD-proficient HT-29 cell line, DZQ and MeDZQ were found to alkylate both 5'-(A/T)G(C)-3' and 5'-(A/T)A-3' sequences. This is consistent with the nucleotide preferences observed when DZQ and MeDZQ are activated by purified DTD to reactive metabolites capable of alkylating DNA in vitro (C-S. Lee, J. A. Hartley, M. D. Berardini, J. Butler, D. Siegel, D. Ross, and N. W. Gibson. Biochemistry, 31: 3019-3025, 1992). Surprisingly in the DTD-deficient BE cell line a pattern of alkylation induced by DZQ and MeDZQ similar to that observed in the DTD-proficient HT-29 cells was observed. This suggests that reductive enzymes other than DTD can be involved in activating DZQ and MeDZQ to DNA-reactive species in vivo.

Alkylation↗
PubMed1994

Slow repair of pyrimidine dimers at p53 mutation hotspots in skin cancer.

Ultraviolet light has been linked with the development of human skin cancers. Such cancers often exhibit mutations in the p53 tumor suppressor gene. Ligation-mediated polymerase chain reaction was used to analyze at nucleotide resolution the repair of cyclobutane pyrimidine dimers along the p53 gene in ultraviolet-irradiated human fibroblasts. Repair rates at individual nucleotides were highly variable and sequence-dependent. Slow repair was seen at seven of eight positions frequently mutated in skin cancer, suggesting that repair efficiency may strongly contribute to the mutation spectrum in a cancer-associated gene.

Cells, Cultured↗
PubMed1993

Detection of DNA adducts at the DNA sequence level by ligation-mediated PCR.

Many carcinogens and mutagens interact with DNA to form specific adducts. Base-specificity and sequence-specificity of adduct formation has been analyzed previously with cloned, end-labelled DNA fragments. However, the distribution of adducts along a mammalian chromosome may be modulated by chromatin structure and could be different from that in naked plasmid DNA. Recently, a method has been developed that utilizes the sensitivity of the polymerase chain reaction (PCR) to detect DNA adducts at the DNA sequence level in mammalian cells. The sequence position of adducts can be mapped whenever it is possible to convert the adduct, either chemically or enzymatically, into a DNA strand break with a 5'-phosphate group. Fragments containing these ligatable breaks are amplified in a single-sided, ligation-mediated PCR reaction. We have used ligation-mediated PCR for detection of alkylguanine adducts and UV-induced cyclobutane pyrimidine dimers and (6-4) photoproducts. We discuss the sensitivity of the method, its limitations, and its potential for mapping other DNA adducts at the DNA sequence level in mammalian cells.

Animals↗
PubMed1993

In vivo protein-DNA interactions at the c-jun promoter: preformed complexes mediate the UV response.

Irradiation of cells with UV light triggers a genetic response, called the UV response, which results in induction of a set of genes containing AP-1-binding sites. The c-jun gene itself, which codes for AP-1-binding activity, is strongly (> 100-fold) and rapidly activated by UV. The UV induction of c-jun is mediated by two UV response elements consisting of AP-1-like sequences within its 5' control region. We have analyzed protein-DNA interactions in vivo at the c-jun promoter in noninduced and UV-irradiated HeLa cells. In vivo footprint analysis was performed by using dimethyl sulfate on intact cells and DNase I on lysolecithihin-permeabilized cells in conjunction with ligation-mediated polymerase chain reaction to cover about 450 bp of the c-jun promoter, including the transcription start sites. We find that this region does not contain methylated cytosines and is thus a typical CpG island. In uninduced cells, in vivo protein-DNA interactions were localized to an AP-1-like sequence (nucleotides [nt] -71 to -64), a CCAAT box element (nt -91 to -87), two SP1 sequences (nt -115 to -110 and -123 to -118), a nuclear factor jun site (nt -140 to -132), and a second AP-1-like sequence (nt -190 to -183). These results indicate that complex protein-DNA interactions exist at the c-jun promoter prior to induction by an external stimulus. Surprisingly, after stimulation of c-jun expression by UV irradiation, all in vivo protein-DNA contacts remained essentially unchanged, including the two UV response elements located at the AP-1-like sequences. The UV-induced signalling cascade leads to phosphorylation of c-Jun on serines 63 and 73 (Y. Devary, R.A. Gottlieb, T. Smeal, and M. Karin, Cell 71:1081-1091, 1992). Taken together, these data suggest that modification of the transactivating domain of DNA-bound c-Jun or a closely related factor may trigger the rapid induction of the c-jun gene.

Base Sequence↗
PubMed1993

The distribution of UV photoproducts along the human p53 gene and its relation to mutations in skin cancer.

Mutations in the p53 gene have been found in a large proportion of human skin cancers. These mutations show the same characteristics as mutations induced by UV light in experimental systems. To establish correlations between formation of DNA adducts by a known carcinogen and incidence of mutations within a specific human gene, we have investigated the formation of UV-induced photoproducts along exons 5-9 of the p53 gene after UV irradiation of human cells. The two major types of DNA photoproducts, cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts [(6-4) photoproducts], were mapped at the DNA sequence level by strand cleavage at the sites of photoproducts. This was followed by ligation-mediated polymerase chain reaction (LMPCR) to amplify gene-specific fragments. In human skin cancers, mutations were most frequently found at codons 151/152, 245, 248, 278 and 286 of the p53 gene. The frequency of UV photoproducts is particularly high at codon 286, which is within a run of 12 adjacent pyrimidines. High levels of both photoproducts were also seen at codons 151 and 278. However, UV-induced DNA adducts are barely detectable at codons 245 and 248, which are mutation hotspots also for internal malignancies. At these positions, the frequency of photoproducts is much lower than at surrounding dipyrimidine sequences. These findings have some implications on molecular mechanisms of mutagenesis in the human genome.

Base Sequence↗