Nucleosomes on linear duplex DNA allow homologous pairing but prevent strand exchange promoted by RecA protein.
To understand the molecular basis of gene targeting, we have studied interactions of nucleoprotein filaments comprised of single-stranded DNA and RecA protein with chromatin templates reconstituted from linear duplex DNA and histones. We observed that for the chromatin templates with histone/DNA mass ratios of 0.8 and 1.6, the efficiency of homologous pairing was indistinguishable from that of naked duplex DNA but strand exchange was repressed. In contrast, the chromatin templates with a histone/DNA mass ratio of 9.0 supported neither homologous pairing nor strand exchange. The addition of histone H1, in stoichiometric amounts, to chromatin templates quells homologous pairing. The pairing of chromatin templates with nucleoprotein filaments of RecA protein-single-stranded DNA proceeded without the production of detectable networks of DNA, suggesting that coaggregates are unlikely to be the intermediates in homologous pairing. The application of these observations to strategies for gene targeting and their implications for models of genetic recombination are discussed.
Ramdas
J
J
Department of Biochemistry, Indian Institute of Science, Bangalore.
Mythili
E
E
Muniyappa
K
K
D000818:Animals; D002843:Chromatin / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D009707:Nucleosomes / Q000378:metabolism*; D010957:Plasmids; D051381:Rats; D011938:Rec A Recombinases / Q000378:metabolism*; D015183:Restriction Mapping; D013698:Templates, Genetic
D002843:Chromatin; D004277:DNA, Single-Stranded; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D006657:Histones; D009707:Nucleosomes; D011938:Rec A Recombinases