Abstract
RecAc38 protein, a chimeric RecA protein of Escherichia coli and Pseudomonas aeruginosa, is proficient in the renaturation from complementary single strands. However, RecAc38 protein showed a significant deficiency in promoting homologous pairing of single-stranded DNA and double-stranded DNA. RecAc38 protein was able to remove the secondary structure of single-stranded DNA, the first step of homologous pairing, at a slightly reduced rate. RecAc38 protein-single-stranded DNA-complex (presynaptic complex) was found to be deficient in the sequence-independent binding to double-stranded DNA which is a step in the search for the homology. On the other hand, once unwinding was initiated, RecAc38 protein was able to propagate the unwinding of the double helix at the same extent as wild-type RecA protein. These defects and the proficiency of RecAc38 protein are explained by a model showing that RecA protein has three distinct DNA strand-binding sites (a site for the primary binding to single-stranded DNA, a site for the binding to a strand of second single- or double-stranded DNA, and a site required for the binding to the other strand of the double-stranded DNA) and that RecAc38 protein has a defect in the third site.
Highlights
Esch- “presynaptic complex”) and at the same timeremoves the secerichia coli and Pseudomonas aeruginosa, is proficient ondary structure in the single-strandedDNA (Kahn and Radin the renaturation from complementary single stranddsi.ng, 1984; Tsang et al, 1985b)
We found that one of the chimera proteins, RecAc38 protein, is defective in some modes of binding to DNA molecules, and this finding provided an insight into DNA binding and its specificities
(Shibata et al, 1982a, 1982b)."hen, we examined homologous pairing by RecAc38 protein with linear double-stranded DNA
Summary
(Received forpublication, May 27, 1993, and in revised form, September 28, 1993). Hitoshi KurumizakaSBl, Shukuko IkawaS, Tomoatsu Ikeyall, Tomoko Ogawall, and Takehiko ShibataS§**. Homologous pairing consists of two reaction phases, the pre- stranded DNA was nucleated by various means, RecA protein synaptic phase and synapsis (Raddinetg al., 1983).In the pre- binds to double-strandedDNA and propagates thuenwinding synapticphase, RecA proteinbinds cooperatively to single- of the double helix in an ATP-dependent and single-stranded stranded DNA to form a nucleoprotein filament 198713).The means to nucleate the bindingof RecA protein to double-stranded DNA include (i)the formationof homologous joints as described above(Ohtani et al.,1982),(ii)the formation Both ternary complex and coaggregate indicate essentiallythe same three-componentcomplex (withrespect to macromolecules), but the principle for assayingis different between themt;he former is detected by means of a filter binding assay and the latter by means of a centrifugation assay (Shibataet al., 1979a; Tsanget al., 1985a). We found that one of the chimera proteins, RecAc38 protein, is defective in some modes of binding to DNA molecules, and this finding provided an insight into DNA binding and its specificities
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