Transposon-mediated site-specific recombination: Identification of three binding sites for resolvase at the res sites of γδ and Tn 3

Grindley, Nigel D. F.; Lauth, M.R.; Wells, Rebecca G.; Wityk, R.J.; Salvo, Joseph J.; Reed, Randall R.

doi:10.1016/0092-8674(82)90007-1

Cited by 148 publications

(68 citation statements)

References 19 publications

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“…The data presented here on Tn3 resolvase extend the observations of Grindley et al (1982) on -y6 resolvase and underline the similarity of these related site-specific recombination systems. All sequences necessary for Tn3 res function lie within the intercistronic region between tnpA and tnpR and are probably contained within the 120-bp region protected from DNase digestion by resolvase binding.…”

Section: Discussionsupporting

confidence: 60%

“…As in other characterized sitespecific recombination systems, the recombination enzyme, resolvase, is encoded by a gene (tnpR) adjacent to the recombination site (res). Tn3 resolvase and res are both similar to and interchangeable with the site-specific recombination ' determinants specified by the related transposable element -y6 (Kostriken et al, 1981;Reed, 1981a;Grindley et al, 1982;Kitts et al, 1982b). Both resolvases act rapidly and efficiently on directly repeated res regions in the same molecule but appear to act poorly in the reverse fusion reaction and on inverted substrates in the same molecule (Chiang and Clowes, 1982;Reed and Grindley, 1981).…”

Section: Introductionmentioning

confidence: 99%

“…(Figure 2; see also Figure 8 of Grindley et al, 1982). There seems to be no preferable resolvase binding to any of these sites when protein is limiting, and ethidium bromide (0.1-20 Atg/ml) did not affect the protection pattern.…”

Section: Introductionmentioning

confidence: 99%

“…Recently, Grindley et al (1982) reported the res sequences within y6 and Tn3 to which -yb resolvase binds. (Figure 2; see also Figure 8 of Grindley et al, 1982).…”

Section: Introductionmentioning

confidence: 99%

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Transposon-encoded site-specific recombination: nature of the Tn3 DNA sequences which constitute the recombination site res.

Kitts¹,

Symington²,

Dyson³

et al. 1983

The EMBO Journal

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The tnpR gene of transposon Tn3 encodes a site-specific recombination enzyme that acts at res, a DNA region adjacent to tnpR, to convert co-integrate intermediates of interreplicon transposition to the normal transposition end-products. We have used two complementary approaches to study the nature of the Tn3 recombination region, res. Firstly, the DNA-binding sites for tnpR protein were determined in DNase I protection experiments. These identified a 120-bp region between the tnpA and tnpR genes that can be subdivided into three separate protein-binding sites. Genetic dissection experiments indicate that few, if any, other sequences in addition to this 120-bp region are required for res function. Moreover, we have shown that the two directly repeated res regions within a molecule are unequal partners in the recombination reaction: a truncated res region, which is unable to recombine with a second identical res region, can recombine efficiently with an intact res region. This demonstration, along with the observation that tnpR/res recombination acts efficiently on directly repeated res regions within a molecule but inefficiently both on inverted res regions in the same molecule and in the fusion reaction between res regions in different molecules, leads us to propose that onedimensional diffusion (tracking) of tnpR protein along DNA is used to locate an initial res region, and then to bring a second directly repeated res region into a position that allows recombination between the res regions.

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Section: Discussionsupporting

confidence: 60%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…Recently, Grindley et al (1982) reported the res sequences within y6 and Tn3 to which -yb resolvase binds. (Figure 2; see also Figure 8 of Grindley et al, 1982).…”

Section: Introductionmentioning

confidence: 99%

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Transposon-encoded site-specific recombination: nature of the Tn3 DNA sequences which constitute the recombination site res.

Kitts¹,

Symington²,

Dyson³

et al. 1983

The EMBO Journal

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“…Tn3 (9,24), also catalyze recombination events between specific sites containing small regions of homology. The integration and excision of lambdoid phages (15) and the recombination events seen with the phage P1 lox-cre system (10) are also mediated through short homologous sequences.…”

mentioning

confidence: 99%

The minimum amount of homology required for homologous recombination in mammalian cells.

Rubnitz¹,

Subramani²

1984

Mol. Cell. Biol.

247

168

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Although DNA sequence homology is believed to be a prerequisite for homologous recombination events in procaryotes and eucaryotes, no systematic study has been done on the minimum amount of homology required for homologous recombination in mammalian cells. We have used simian virus 40-pBR322 hybrid plasmids constructed in vitro as substrates to Tn3 (9, 24), also catalyze recombination events between specific sites containing small regions of homology. The integration and excision of lambdoid phages (15) and the recombination events seen with the phage P1 lox-cre system (10) are also mediated through short homologous sequences. In eucaryotes, the FLP protein of the yeast 2,u plasmid catalyzes site-specific recombination between inverted repeats present on the plasmid (4). Other demonstrations of the importance of short homologies include nonhomologous recombinations lacking site specificity. Nonhomologous recombination between phage A DNA and pBR322 DNA in Escherichia coli may involve 10 to 13 base pairs (bp) of homology (18), whereas deletions in E. coli may be formed by recombination between homologous regions of 6 to 17 bp (1).Because sequence homology is such an important feature of recombination, several investigators have studied the effects of decreasing the length of homology available for specific recombination events. First, Singer et al. (30) determined that a minimum of 50 bp of homology is required by the major recombination pathway of bacteriophage T4. More recently, Gonda and Radding (8) found that E. coli RecA protein efficiently paired molecules that shared 151 bp of homology but failed to pair molecules in which homology was limited to 30 bp. In the present work, we have used previously described pBR322-simian virus 40 (SV40) hybrid plasmids (34) to investigate the minimum amount of homology required for recombination in monkey cells. After transfection of these plasmids into permissive cells, homologous recombination events leading to the formation of infectious SV40 virions can be easily scored by a plaque assay (see below). By progressively decreasing the length of homology from 5,243 to 0 bp, we have measured recombination frequency as a function of the length of homology. Our results show that the steepest drop in recombination frequency occurred between 163 and 214 bp, with lower levels of recombination occurring when there was only 14 bp of homology.

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Site‐specific recombination systems for the genetic manipulation of eukaryotic genomes

Thomson

2006

Genesis

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Site-specific recombination systems, such as the bacteriophage Cre-lox and yeast FLP-FRT systems, have become valuable tools for the rearrangement of DNA in higher eukaryotes. As a first step to expanding the repertoire of recombination tools, we screened recombination systems derived from the resolvase/invertase family for site-specific recombinase activity in the fission yeast Schizosaccharomyces pombe. Here, we report that seven recombination systems, four from the small serine resolvase subfamily (CinH, ParA, Tn1721, and Tn5053) and three from the large serine resolvase subfamily (Bxb1, TP901-1, and U153), can catalyze site-specific deletion in S. pombe. Those from the large serine resolvase subfamily were also capable of site-specific integration and inversion. In all cases, the recombination events were precise. Functional operation of these recombination systems in the fission yeast holds promise that they may be further developed as recombination tools for the site-specific rearrangement of plant and animal genomes.

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Transposon-mediated site-specific recombination: Identification of three binding sites for resolvase at the res sites of γδ and Tn 3

Cited by 148 publications

References 19 publications

Transposon-encoded site-specific recombination: nature of the Tn3 DNA sequences which constitute the recombination site res.

Transposon-encoded site-specific recombination: nature of the Tn3 DNA sequences which constitute the recombination site res.

The minimum amount of homology required for homologous recombination in mammalian cells.

Site‐specific recombination systems for the genetic manipulation of eukaryotic genomes

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