The role of theloxPspacer region in PI site-specific recombination

Abstract

The lox-Cre site-specific recombination system of bacteriophage P1 is comprised of a site on the DNA where recombination occurs called loxP, and a protein, Cre, which mediates the reaction. The loxP site is 34 base pairs (bp) in length and consists of two 13 bp inverted repeats separated by an 8 bp spacer region. Previously it has been shown that the cleavage and strand exchange of recombining loxP sites occurs within this spacer region. We report here an analysis of various base substitution mutations within the spacer region of loxP, and conclude the following: Homology is a requirement for efficient recombination between recombining loxP sites. There is at least one position within the spacer where a base change drastically reduces recombination even when there is homology between the two recombining loxP sites. When two loxP sites containing symmetric spacer regions undergo Cre-mediated recombination in vitro, the DNA between the sites undergoes both excision and inversion with equal frequency.

Defining the Functional Boundaries of the Gata2 Locus by Rescue with a Linked Bacterial Artificial Chromosome Transgene

“…1, A and B). To fuse the two BACs precisely in register, we employed cre-mediated homologous recombination to exploit the documented differential recombination efficiency between homologous versus heterologous loxP sequences (24). A single loxP514 site (yellow arrow, Fig.…”

Analysis of spacer regions derived from intramolecular recombination between heterologous loxP sites

“…When two loxP sites are placed in the same orientation on the same DNA molecule, intramolecular recombination results in the excision of the DNA segment intervening two loxP sites [3,4]. A series of plasmids for the detection of recombination event was constructed by employing wt loxP and five mutant loxP sites including M2, M3, M7, M11, and 2272 that contain the altered bases within their spacer region ( Fig.…”

“…An asymmetric spacer region determines the orientation of a loxP site and directionality of recombination events, either excision or inversion. Recombination between two loxP sites placed in the same orientation on the same DNA molecule results in the excision of the DNA segment intervening two loxP sites, whereas the inversion of DNA segment in recombination between the opposite-oriented two loxP sites [3,4]. With respect to the cleavage in loxP, cleaved sites are apart from 6 bp (core or exchange region) within a 8 bp spacer region.…”

“…The resulting recombinant products become to have the bases different from the parental spacers at position 4 0 and 4. Sequence homology within 6 bp cores is required for efficient strand exchange but it does not seem to be strict requisition because the productive recombination events were actually observed between loxP sites with heterologous spacers in a variety of degree [3,[5][6][7].The mutant loxP sites with the altered spacer have been extensively studied to elucidate the role of nucleotide sequence of spacer region or to identify the one that undergoes recombination with the same mutant loxP but not other mutant loxP or wild type loxP site (wt loxP) [5,6,8]. These studies have examined intramolecular recombination efficiency between mutant loxPs bearing altered spacers as well as between wt loxP and mutant loxP, without any analyses for spacer sequence left in individual products after recombination.…”

“…The resulting recombinant products become to have the bases different from the parental spacers at position 4 0 and 4. Sequence homology within 6 bp cores is required for efficient strand exchange but it does not seem to be strict requisition because the productive recombination events were actually observed between loxP sites with heterologous spacers in a variety of degree [3,[5][6][7].…”