Enhanced CPT sensitivity of yeast cells and selective relaxation of Ga14 motif-containing DNA by novel Gal4-topoisomerase I fusion proteins.
Human topoisomerase I-B (Top1) efficiently relaxes DNA supercoils during basic cellular processes, and can be transformed into a DNA-damaging agent by antitumour drugs, enzyme mutations and DNA lesions. Here, we describe Gal4-Top1 chimeric proteins (GalTop) with an N-terminal truncation of Top1, and mutations of the Gal4 Zn-cluster and/or Top1 domains that impair their respective DNA-binding activities. Expression levels of chimeras were similar in yeast cells, however, GalTop conferred an increased CPT sensitivity to RAD52- yeast cells as compared to a GalTop with mutations of the Gal4 domain, showing that a functional Gal4 domain can alter in vivo functions of Top1. In vitro enzyme activity was tested with a DNA relaxation assay using negatively supercoiled plasmids with 0 to 5 Gal4 consensus motifs. Only GalTop with a functional Gal4 domain could direct DNA relaxation activity of Top1 specifically to DNA molecules containing Gal4 motifs. By using a substrate competition assay, we could demonstrate that the Gal4-anchored Top1 remains functional and efficiently relax DNA substrates in cis. The enhanced CPT sensitivity of GalTop in yeast cells may then be due to alterations of the chromatin-binding activity of Top1. The GalTop chimeras may indeed mimic a normal mechanism by which Top1 is recruited to chromatin sites in living cells. Such hybrid Top1s may be helpful in further dissecting enzyme functions, and constitute a prototype of a site-specific DNA cutter endowed with high cell lethality.
Alessandri
Marco
M
Alma Mater Studiorum-University of Bologna, G. Moruzzi Department of Biochemistry, via S. Giacomo 11, 40126 Bologna, Italy.
Beretta
Giovanni L
GL
Ferretti
Edmondo
E
Mancia
Annalaura
A
Khobta
Andriy
A
Capranico
Giovanni
G
D020816:Amino Acid Motifs; D002166:Camptothecin / Q000494:pharmacology*; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004271:DNA, Fungal / Q000737:chemistry* / Q000187:drug effects* / Q000235:genetics; D004278:DNA, Superhelical / Q000737:chemistry / Q000187:drug effects / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004355:Drug Stability; D006801:Humans; D066298:In Vitro Techniques; D009690:Nucleic Acid Conformation / Q000187:drug effects; D051721:Rad52 DNA Repair and Recombination Protein; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012965:Sodium Chloride; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004271:DNA, Fungal; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; C497197:RAD52 protein, S cerevisiae; D051721:Rad52 DNA Repair and Recombination Protein; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D012965:Sodium Chloride; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin