Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A novel assay to detect strand-specific DNA repair after cellular exposure to cisplatin at IC50 levels, is used to measure rapid repair in the divergent upstream gene (DUG), a human MutS homolog, and in the bidirectional promoter for dihydrofolate reductase gene (DHFR) and the contiguous upstream DUG. Single-stranded DNA capable of hybridizing to gene-specific probes is generated enzymatically by the 3'-5' exonuclease activity of T4 DNA polymerase. The presence of cisplatin lesions inhibit the exonucleolytic activity of T4 DNA polymerase and block the formation of single-stranded DNA. This decreases the amount of complementary sequence produced when assayed by gene-specific probe hybridization. With the progression of repair, increasing quantities of single-stranded DNA become available for probe hybridization. This assay was applied to human A2780 ovarian carcinoma cells treated with cisplatin at the beginning of G1 phase. A dose-response experiment showed that the assay was applicable down to cisplatin concentrations of 2.5 microM. To assay for strand-specific gene repair, the synchronized cells were treated with cisplatin and then allowed time to repair in drug-free medium. Extensive removal of cisplatin lesions after 2 hr of cellular repair during early G1 phase in the DUG and the DUG/DHFR promoter was measured, with no evidence of repair in the unexpressed delta-globin gene. The extent of preferential DNA repair was much more distinct than has been observed previously at high-drug dosage in asynchronous cells.
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PMID:Rapid gene-specific repair of cisplatin lesions at the human DUG/DHFR locus comprising the divergent upstream gene and dihydrofolate reductase gene during early G1 phase of the cell cycle assayed by using the exonucleolytic activity of T4 DNA polymerase. 797 95

The level and fate of hMSH3 (human MutS homolog 3) were examined in the promyelocytic leukemia cell line HL-60 and its methotrexate-resistant derivative HL-60R, which is drug resistant by virtue of an amplification event that spans the dihydrofolate reductase (DHFR) and MSH3 genes. Nuclear extracts from HL-60 and HL-60R cells were subjected to an identical, rapid purification protocol that efficiently captures heterodimeric hMutSalpha (hMSH2. hMSH6) and hMutSbeta (hMSH2.hMSH3). In HL-60 extracts the hMutSalpha to hMutSbeta ratio is roughly 6:1, whereas in methotrexate-resistant HL-60R cells the ratio is less than 1:100, due to overproduction of hMSH3 and heterodimer formation of this protein with virtually all the nuclear hMSH2. This shift is associated with marked reduction in the efficiency of base-base mismatch and hypermutability at the hypoxanthine phosphoribosyltransferase (HPRT) locus. Purified hMutSalpha and hMutSbeta display partial overlap in mismatch repair specificity: both participate in repair of a dinucleotide insertion-deletion heterology, but only hMutSalpha restores base-base mismatch repair to extracts of HL-60R cells or hMSH2-deficient LoVo colorectal tumor cells.
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PMID:DHFR/MSH3 amplification in methotrexate-resistant cells alters the hMutSalpha/hMutSbeta ratio and reduces the efficiency of base-base mismatch repair. 929 77