Abstract
Genetic recombination occurring in wild type Escherichia coli is stimulated at DNA sequences known as χ sites, 5′-GCTGGTGG-3′. In vitro, homologous pairing between duplex DNA substrates dependent upon the RecA, RecBCD, and SSB proteins is stimulated by the presence of a χ sequence in the donor linear double-stranded DNA. We show that this stimulation is due to two factors: 1) the enhanced production of χ-specific single-stranded DNA fragments and 2) their preferential use in the RecA protein-promoted pairing step. Furthermore, under conditions of limiting Mg2+ concentration, joint molecule formation does not occur, even though DNA unwinding and χ-specific single-stranded DNA fragment production are observed. Also, under these conditions, χ-specific fragments derived from both the upstream and downstream regions of the DNA strand containing χ and from cleavage of the non-χ-containing DNA strand are detected. Finally, the behavior of mutant RecBCD enzymes (RecBC*D and RecBCD‡) in this in vitro reaction is shown to parallel their in vivo phenotypes with respect to χ stimulation of recombination. Thus we suggest that, in addition to its ability to regulate the degradative activities of RecBCD enzyme, χ itself may be a preferred site for initiation of homologous pairing in this concerted process.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 16360-16370 |
| Number of pages | 11 |
| Journal | Journal of Biological Chemistry |
| Volume | 270 |
| Issue number | 27 |
| State | Published - Jul 7 1995 |
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ASJC Scopus subject areas
- Biochemistry
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Role of the Escherichia coli recombination hotspot, χ, in RecABCD-dependent homologous pairing. / Dixon, Dan A.; Kowalczykowski, Stephen C.
In: Journal of Biological Chemistry, Vol. 270, No. 27, 07.07.1995, p. 16360-16370.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Role of the Escherichia coli recombination hotspot, χ, in RecABCD-dependent homologous pairing
AU - Dixon, Dan A.
AU - Kowalczykowski, Stephen C.
PY - 1995/7/7
Y1 - 1995/7/7
N2 - Genetic recombination occurring in wild type Escherichia coli is stimulated at DNA sequences known as χ sites, 5′-GCTGGTGG-3′. In vitro, homologous pairing between duplex DNA substrates dependent upon the RecA, RecBCD, and SSB proteins is stimulated by the presence of a χ sequence in the donor linear double-stranded DNA. We show that this stimulation is due to two factors: 1) the enhanced production of χ-specific single-stranded DNA fragments and 2) their preferential use in the RecA protein-promoted pairing step. Furthermore, under conditions of limiting Mg2+ concentration, joint molecule formation does not occur, even though DNA unwinding and χ-specific single-stranded DNA fragment production are observed. Also, under these conditions, χ-specific fragments derived from both the upstream and downstream regions of the DNA strand containing χ and from cleavage of the non-χ-containing DNA strand are detected. Finally, the behavior of mutant RecBCD enzymes (RecBC*D and RecBCD‡) in this in vitro reaction is shown to parallel their in vivo phenotypes with respect to χ stimulation of recombination. Thus we suggest that, in addition to its ability to regulate the degradative activities of RecBCD enzyme, χ itself may be a preferred site for initiation of homologous pairing in this concerted process.
AB - Genetic recombination occurring in wild type Escherichia coli is stimulated at DNA sequences known as χ sites, 5′-GCTGGTGG-3′. In vitro, homologous pairing between duplex DNA substrates dependent upon the RecA, RecBCD, and SSB proteins is stimulated by the presence of a χ sequence in the donor linear double-stranded DNA. We show that this stimulation is due to two factors: 1) the enhanced production of χ-specific single-stranded DNA fragments and 2) their preferential use in the RecA protein-promoted pairing step. Furthermore, under conditions of limiting Mg2+ concentration, joint molecule formation does not occur, even though DNA unwinding and χ-specific single-stranded DNA fragment production are observed. Also, under these conditions, χ-specific fragments derived from both the upstream and downstream regions of the DNA strand containing χ and from cleavage of the non-χ-containing DNA strand are detected. Finally, the behavior of mutant RecBCD enzymes (RecBC*D and RecBCD‡) in this in vitro reaction is shown to parallel their in vivo phenotypes with respect to χ stimulation of recombination. Thus we suggest that, in addition to its ability to regulate the degradative activities of RecBCD enzyme, χ itself may be a preferred site for initiation of homologous pairing in this concerted process.
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M3 - Article
C2 - 7608206
AN - SCOPUS:0029038312
VL - 270
SP - 16360
EP - 16370
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 27
ER -