DNA-binding activities of Hop1 protein, a synaptonemal complex component from Saccharomyces cerevisiae.
The meiosis-specific HOP1 gene is important both for crossing over between homologs and for production of viable spores. hop1 diploids fail to assemble synaptonemal complex (SC), which normally provides the framework for meiotic synapsis. Immunochemical methods have shown that the 70-kDa HOP1 product is a component of the SC. To assess its molecular function, we have purified Hop1 protein to homogeneity and shown that it forms dimers and higher oligomers in solution. Consistent with the zinc-finger motif in its sequence, the purified protein contained about 1 mol equivalent of zinc whereas mutant protein lacking a conserved cysteine within this motif did not. Electrophoretic gel mobility shift assays with different forms of M13 DNA showed that Hop1 binds more readily to linear duplex DNA and negatively superhelical DNA than to nicked circular duplex DNA and even more weakly to single-stranded DNA. Linear duplex DNA binding was enhanced by the addition of Zn2+, was stronger for longer DNA fragments, and was saturable to about 55 bp/protein monomer. Competitive inhibition of this binding by added oligonucleotides suggests preferential affinity for G-rich sequences and weaker binding to poly(dA-dT). Nuclear extracts of meiotic cells caused exonucleolytic degradation of linear duplex DNA if the extracts were prepared from hop1 mutants; addition of purified Hop1 conferred protection against this degradation. These findings suggest that Hop1 acts in meiotic synapsis by binding to sites of double-strand break formation and helping to mediate their processing in the pathway to meiotic recombination.
Kironmai
K M
KM
Department of Biochemistry, Indian Institute of Science, Bangalore.
Muniyappa
K
K
Friedman
D B
DB
Hollingsworth
N M
NM
Byers
B
B
D002413:Cations, Divalent; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005092:Exonucleases / Q000378:metabolism; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D008274:Magnesium; D009690:Nucleic Acid Conformation; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship; D013573:Synaptonemal Complex; D015032:Zinc
D002413:Cations, Divalent; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C086672:HOP1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D005092:Exonucleases; D008274:Magnesium; D015032:Zinc