FORM OF THE DNA SUBSTRATE REQUIRED FOR EXCISIVE RECOMBINATION OF BACTERIOPHAGE-LAMBDA

被引:30
作者
ABREMSKI, K
GOTTESMAN, S
机构
[1] Laboratory of Molecular Biology National Cancer Institute National Institutes of Health Bethesda
关键词
D O I
10.1016/0022-2836(79)90012-3
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We have studied the excision reaction of bacteriophage lambda, both in vivo and in vitro, using as a substrate a λatt2(L × R) phage carrying both the right and left-hand prophage attachment sites. Int and Xis are provided by induction of the heat-inducible defective prophage, λc1857 ΔH1. After a brief induction (5 min) of these cells, excisive recombination is blocked in the presence of the DNA gyrase inhibitor, coumermycin. However, after a longer induction (greater than 30 min) excisive recombination occurs efficiently under conditions where λ integrative recombination is inhibited by coumermycin. In such extensively induced coumermycin-treated cells, infecting λatt2(L × R) DNA is not supercoiled, and recombinants are found among the relaxed covalently closed circular DNA. In vitro, starting with a hydrogen-bonded λatt2 DNA substrate, excision is insensitive to high concentrations of coumermycin and novobiocin. To study the DNA substrate requirements for excisive recombination in more detail, we have developed a restriction fragment assay for excisive recombination. With this assay, we demonstrate that supercoiled, hydrogen-bonded, and linear λatt2 DNA molecules are all efficient substrates in the in vitro excision reaction. Spermidine is required but ATP and Mg2+ are not. We conclude that supercoiling is not an absolute requirement for site-specific recombination of λ. © 1979.
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页码:637 / 649
页数:13
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