Structure of Cre recombinase complexed with DNA in a site-specific recombination synapse

Guo, Feng; Gopaul, Deshmukh N.; Duyne, Gregory D. Van

doi:10.1038/37925

Cited by 548 publications

(706 citation statements)

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“…2,4 Replacement of E319 by any of several amino acids (including glycine), whose only common element is loss of a negative charge, increases recombination of HK022 sites. 22 These considerations suggest that E319 does not directly contact core site DNA, but instead affects site recognition indirectly, perhaps through interaction with residues that themselves contact DNA.…”

Section: Discussionmentioning

confidence: 99%

Analysis of Insertion into Secondary Attachment Sites by Phage λ and by int Mutants with Altered Recombination Specificity

Rutkai

Dorgai

Sirot

et al. 2003

Journal of Molecular Biology

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When phage l lysogenizes a cell that lacks the primary bacterial attachment site, integrase catalyzes insertion of the phage chromosome into one of many secondary sites. Here, we characterize the secondary sites that are preferred by wild-type l and by l int mutants with altered insertion specificity. The sequences of these secondary sites resembled that of the primary site: they contained two imperfect inverted repeats flanking a short spacer. The imperfect inverted repeats of the primary site bind integrase, while the 7 bp spacer, or overlap region, swaps strands with a complementary sequence in the phage attachment site during recombination. We found substantial sequence conservation in the imperfect inverted repeats of secondary sites, and nearly perfect conservation in the leftmost three bases of the overlap region. By contrast, the rightmost bases of the overlap region were much more variable. A phage with an altered overlap region preferred to insert into secondary sites with the corresponding bases. We suggest that this difference between the left and right segments is a result of the defined order of strand exchanges during integrase-promoted recombination. This suggestion accounts for the unexpected segregation pattern of the overlap region observed after insertion into several secondary sites. Some of the altered specificity int mutants differed from wild-type in secondary site preference, but we were unable to identify simple sequence motifs that account for these differences. We propose that insertion into secondary sites is a step in the evolutionary change of phage insertion specificity and present a model of how this might occur.Published by Elsevier Science Ltd

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Section: Discussionmentioning

confidence: 99%

Analysis of Insertion into Secondary Attachment Sites by Phage λ and by int Mutants with Altered Recombination Specificity

Rutkai

Dorgai

Sirot

et al. 2003

Journal of Molecular Biology

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“…The first issue is the size of deletions: can Cre induce efficient chromosomal recombination over large genetic distances? To cause recombination, two Cre monomers are bound to the two 13 bp inverted repeats of a single lox site before a tetramer complex is formed (Guo et al, 1997), in which the two lox sites are aligned to enable strand exchange via a Holliday-junction intermediate. It is conceivable that at larger genetic distances, the formation of these tetramer complexes is less likely, thus causing a drop in recombination efficiency.…”

Section: Introductionmentioning

confidence: 99%

Size Does Matter: Cre-mediated Somatic Deletion Efficiency Depends on the Distance Between the Target lox-Sites

et al. 2005

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Cre/lox recombination in vivo has become an important tool to induce chromosomal rearrangements like deletions. Using a combination of Ds transposition and Cre/lox recombination in two independent experiments on chromosomes 6 and 7 of tomato, two sets of somatic deletions up to a size of 200 kb were obtained. The efficiency of somatic deletion decreased with increasing deletion size. The largest germinally transmitted deletion had a size of only 55 kb. The results show that Cre-mediated deletion in somatic cells is less efficient when the lox sites are separated over larger distances. A further drop of the deletion efficiency after germinal transmission of the larger deletions can be explained by the probable loss of genes that are of vital importance to gametophyte function. Plasmid rescue of an 8.4 kb circularised deleted DNA showed that the Cre-mediated deletion takes place in tomato as expected. Since the circular Cre-deleted DNA could only be PCR amplified in plant cells where the deletion was not complete, the double-stranded DNA circle is assumed to be instable.

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“…The question of how Cre specifically recognizes its target DNA is not solved in detail yet, although recently the structure of this recombinase bound to its target sequence has been reported to 2.4 Å resolution (18). The analysis of the crystal * This work was supported by the German Israeli Foundation.…”

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confidence: 99%

“…structure with respect to protein function focused on the active sites of the synaptic complex and on the mechanism of Cre-loxP site-specific recombination and less on the target recognition specificity of the protein (18). The crystals of the catalytic cores of HP1 and integrase (19,20), or the crystal of XerD (21), give no final answer to the question of how these proteins specifically recognize their target sequences because these proteins had been crystallized without their target DNA.…”

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confidence: 99%

Cre Mutants with Altered DNA Binding Properties

Hartung

Kisters‐Woike

1998

Journal of Biological Chemistry

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Structure of Cre recombinase complexed with DNA in a site-specific recombination synapse

Cited by 548 publications

References 1 publication

Analysis of Insertion into Secondary Attachment Sites by Phage λ and by int Mutants with Altered Recombination Specificity

Analysis of Insertion into Secondary Attachment Sites by Phage λ and by int Mutants with Altered Recombination Specificity

Size Does Matter: Cre-mediated Somatic Deletion Efficiency Depends on the Distance Between the Target lox-Sites

Cre Mutants with Altered DNA Binding Properties

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