Analysis of mitochondrial DNA nucleoids in wild-type and a mutant strain of Saccharomyces cerevisiae that lacks the mitochondrial HMG box protein Abf2p.
DNA-protein complexes (nucleoids) are believed to be the segregating unit of mitochondrial DNA (mtDNA) in Saccharomyces cerevisiae. A mitochondrial HMG box protein, Abf2p, is needed for maintenance of mtDNA in cells grown on rich dextrose medium, but is dispensible in glycerol grown cells. As visualized by 4',6'-diamino-2-phenylindole staining, mtDNA nucleoids in mutant cells lacking Abf2p ( delta abf2) are diffuse compared with those in wild-type cells. We have isolated mtDNA nucleoids and characterized two mtDNA-protein complexes, termed NCLDp-2 and NCLDs-2, containing distinct but overlapping sets of polypeptides. This protocol yields similar nucleoid complexes from the delta abf2 mutant, although several proteins appear lacking from NCLDs-2. Segments of mtDNA detected with probes to COXII, VAR1 and ori5 sequences are equally sensitive to DNase I digestion in NCLDs-2 and NCLDp-2 from wild-type cells and from the delta abf2 mutant. However, COXII and VAR1 sequences are 4-to 5-fold more sensitive to DNase I digestion of mtDNA in toluene-permeabilized mitochondria from the delta abf2 mutant than from wild-type cells, but no difference in DNase I sensitivity was detected with the ori5 probe. These results provide a first indication that Abf2p influences differential organization of mtDNA sequences.
Newman
S M
SM
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, 75235, USA.
Zelenaya-Troitskaya
O
O
Perlman
P S
PS
Butow
R A
RA
D004271:DNA, Fungal / Q000032:analysis; D004272:DNA, Mitochondrial / Q000032:analysis*; D004268:DNA-Binding Proteins / Q000032:analysis / Q000502:physiology*; D003850:Deoxyribonuclease I; D003576:Electron Transport Complex IV / Q000235:genetics; D005456:Fluorescent Dyes; D005656:Fungal Proteins / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D007211:Indoles; D008565:Membrane Proteins; D008928:Mitochondria / Q000737:chemistry*; D024101:Mitochondrial Proteins; D010539:Permeability; D018741:Replication Origin / Q000235:genetics; D012269:Ribosomal Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000166:cytology*; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000502:physiology*
C072843:ABF2 protein, S cerevisiae; D004271:DNA, Fungal; D004272:DNA, Mitochondrial; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D005656:Fungal Proteins; D007211:Indoles; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C026383:VAR1 protein, S cerevisiae; C007293:DAPI; D003576:Electron Transport Complex IV; D003850:Deoxyribonuclease I