Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A double-stranded circular DNA having a single nick at a specific site has been photochemically induced to react with 4'-hydroxymethyl-4,5', 8-trimethylpsoralen (HMT) and used as a substrate for nick-translation with Escherichia coli DNA polymerase I holoenzyme. By using the dideoxy chain-terminating sequencing procedure, it was possible to map the termination sites observed on the template that had photoreacted with HMT. These sites occur at nucleotides preceding potential psoralen crosslinking sites. Analysis of DNA products synthesized on templates that had photoreacted under conditions designed to maximize psoralen monoaddition revealed that DNA polymerase I is not stopped by this lesion. Psoralen monoadducts situated on the template strand act only as kinetic attenuators, whereas psoralen monoadducts localized on the nick-translated strand have no effect on the rate of synthesis. These data suggest that psoralen crosslinks are responsible for the lethal effects of psoralen photochemistry in E. coli. Mutagenesis may be associated, however, with the repair replication of psoralen monoadducts by E. coli DNA polymerase I.
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PMID:Termination sites of the in vitro nick-translation reaction on DNA that had photoreacted with psoralen. 622 23

Psoralen-conjugated triple-helix-forming oligonucleotides have been used to generate site-specific mutations within mammalian cells. To investigate factors influencing the efficiency of oligonucleotide-mediated gene targeting, the processing of third-strand-directed psoralen adducts was compared in normal and repair-deficient human cells. An unusually high mutation frequency and an altered mutation pattern were seen in xeroderma pigmentosum variant (XPV) cells compared with normal, xeroderma pigmentosum group A (XPA), and Fanconi anemia cells. In XPV, targeted mutations were produced in the supF reporter gene carried in a simian virus 40 vector at a frequency of 30%, 3-fold above that in normal or Fanconi anemia cells and 6-fold above that in XPA. The mutations generated by targeted psoralen crosslinks and monoadducts in the XPV cells formed a pattern distinct from that in the other three cell lines, with mutations occurring not just at the damaged site but also at adjacent base pairs. Hence, the XPV cells may have an abnormality in trans-lesion bypass synthesis during repair and/or replication, implicating a DNA polymerase or an accessory factor as a basis of the defect in XPV. These results may help to elucidate the repair deficiency in XPV, and they raise the possibility that genetic manipulation via triplex-targeted mutagenesis may be enhanced by modulation of the XPV-associated activity in normal cells.
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PMID:Mutagenesis by third-strand-directed psoralen adducts in repair-deficient human cells: high frequency and altered spectrum in a xeroderma pigmentosum variant. 861 Jan 47

DNA-DNA interstrand cross-links are thought to be important for the cytotoxicity of many chemotherapeutic agents. To study this more definitively, adduct site-specific methods are used to construct a plasmid with a single nitrogen mustard interstrand cross-link (inter-HN2-pTZSV28). Replication efficiency (RE = [colonies from (inter-HN2-pTZSV28)/(control with no cross-link)]) is approximately 0.3 following transformation into Escherichia coli, implying that the cross-link is repaired. The commonly accepted pathway for cross-link repair, which involves both nucleotide excision repair (NER) and recombination, is ruled out since RE is approximately 0.3 in a delta recA strain. Non-RecA-directed recombination such as copy-choice is also unlikely. However, NER is involved since RE was approximately 0.02 in strains deficient in NER. Base excision repair is not important since RE is approximately 0.3 in strains deficient in 3-methyladenine DNA glycosylases I and II, FAPY DNA glycosylase, both known apurinic/apyrimidinic endonucleases, or DNA deoxyribophosphodiesterase. Another hypothetical repair pathway hinging on a 5' --> 3' exonuclease activity is unlikely since RE is approximately 0.3 in cells deficient in either the 5' --> 3' exonuclease activities of DNA polymerase I, exonuclease VII, or RecJ. Thus, aside from NER, it is unclear what else participates in this recombination-independent repair pathway, although a pathway involing NER followed by replicative bypass of the lesion is the current working hypothesis. Psoralen interstrand cross-links appear not to be repairable by this second pathway, which may have implications for the relative cytotoxicity of interstrand cross-links from different agents.
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PMID:Evidence for a recombination-independent pathway for the repair of DNA interstrand cross-links based on a site-specific study with nitrogen mustard. 913