RESUMO
rtt4-1 (regulator of Ty transposition) is a cellular mutation that permits a high level of spontaneous Ty1 retrotransposition in Saccharomyces cerevisiae. The RTT4 gene is allelic with SSL2 (RAD25), which encodes a DNA helicase present in basal transcription (TFIIH) and nucleotide excision repair (NER) complexes. The ssl2-rtt (rtt4-1) mutation stimulates Ty1 retrotransposition, but does not alter Ty1 target site preferences, or increase cDNA or mitotic recombination. In addition to ssl2-rtt, the ssl2-dead and SSL2-1 mutations stimulate Ty1 transposition without altering the level of Ty1 RNA or proteins. However, the level of Ty1 cDNA markedly increases in the ssl2 mutants. Like SSL2, certain mutations in another NER/TFIIH DNA helicase encoded by RAD3 stimulate Ty1 transposition. Although Ssl2p and Rad3p are required for NER, inhibition of Ty1 transposition is independent of Ssl2p and Rad3p NER functions. Our work suggests that NER/TFIIH subunits antagonize Ty1 transposition posttranslationally by inhibiting reverse transcription or destabilizing Ty1 cDNA.
Assuntos
Reparo do DNA , Regulação da Expressão Gênica , Biossíntese de Proteínas , Retroelementos , Proteínas de Saccharomyces cerevisiae , Fatores Associados à Proteína de Ligação a TATA , Fator de Transcrição TFIID , Fatores de Transcrição TFII , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Oxirredutases do Álcool , Alelos , Aminoidrolases , Cromossomos Fúngicos , DNA Helicases/genética , DNA Helicases/metabolismo , Elementos de DNA Transponíveis , DNA Complementar , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Mutagênese , Pirofosfatases , Recombinação Genética , Fator de Transcrição TFIIH , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Semidominant mutations in the PDR1 or PDR3 gene lead to elevated resistance to cycloheximide and oligomycin. PDR1 and PDR3 have been demonstrated to encode zinc cluster transcription factors. Cycloheximide resistance mediated by PDR1 and PDR3 requires the presence of the PDR5 membrane transporter-encoding gene. However, PDR5 is not required for oligomycin resistance. Here, we isolated a gene that is necessary for PDR1- and PDR3-mediated oligomycin resistance. This locus, designated YOR1, causes a dramatic elevation in oligomycin resistance when present in multiple copies. A yor1 strain exhibits oligomycin hypersensitivity relative to an isogenic wild-type strain. In addition, loss of the YOR1 gene blocks the elevation in oligomycin resistance normally conferred by mutant forms of PDR1 or PDR3. The YOR1 gene product is predicted to be a member of the ATP-binding cassette transporter family of membrane proteins. Computer alignment indicates that Yor1p shows striking sequence similarity with multidrug resistance-associated protein, Saccharomyces cerevisiae Ycf1p, and the cystic fibrosis transmembrane conductance regulator. Use of a YOR1-lacZ fusion gene indicates that YOR1 expression is responsive to PDR1 and PDR3. While PDR5 expression is strictly dependent on the presence of PDR1 or PDR3, control of YOR1 expression has a significant PDR1/PDR3-independent component. Taken together, these data indicate that YOR1 provides the link between transcriptional regulation by PDR1 and PDR3 and oligomycin resistance of yeast cells.
