A second site-specific recombination event in the lambdoid bacteriophage HK022

Kolot, Mikhail; Gottfried, Pnina; Yagil, Ezra

doi:10.1007/s004380050332

Cited by 5 publications

(4 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This involved a second round of recombination between the mutant attP substrate and presumably the junction sites on the primary attP´attB recombinant product. Such sequential reactions have been observed in studies of the site-speci®c integration systems of phages HP1 and HK022 (Goodman and Scocca 1989;Hakimi and Scocca 1996;Kolot et al 1996). Recombination between attP and attL or attR has also been reported for lambda (Echols 1970;Guarneros and Echols 1973;Hsu et al 1980).…”

Section: Discussionmentioning

confidence: 67%

Integrative recombination of Lactobacillus delbrueckii bacteriophage mv4: functional analysis of the reaction and structure of the attP site

Auvray

Coddeville²,

Espagno³

et al. 1999

Mol Gen Genet

View full text Add to dashboard Cite

The integrase of the temperate bacteriophage mv4 catalyzes site-specific recombination between the phage attP site and the attB site of the host during lysogenization of Lactobaccillus delbrueckii subsp. bulgaricus. The mv4 integrase also functions in a wide variety of gram-positive bacteria and in Escherichia coli. In this report, in vitro and in vivo recombination assays were developed and the integrase was purified in order to study in greater detail the mv4 attP x attB recombination event. In a cell-free extract of E. coli at 42 degrees C, the mv4 integrase promotes efficient in vitro recombination between a supercoiled attP-containing plasmid and a linear attB fragment. The integrase, which was purified to apparent homogeneity, showed no absolute requirement for accessory factors, unlike the majority of the lambda Int family of recombinases. Deletion derivatives of the attP site were constructed and tested for recombination with the attB site in vitro. A 234-bp DNA fragment containing five scattered putative mv4 Int-binding sites was sufficient for function of the attP site. In contrast to the right arm of attP, most of the left arm could be deleted without drastically reducing the recombination efficiency. In vivo in E. coli, mv4 Int catalyzed recombination in trains between attP and attB sites present on two separate plasmids. This property was used to confirm in vivo the results of the deletion analysis of the attP site performed in vitro.

show abstract

Section: Discussionmentioning

confidence: 67%

Integrative recombination of Lactobacillus delbrueckii bacteriophage mv4: functional analysis of the reaction and structure of the attP site

Auvray

Coddeville²,

Espagno³

et al. 1999

Mol Gen Genet

View full text Add to dashboard Cite

show abstract

“…Plasmid construction pKY113: attP was generated by PCR from pKY110 (Kolot et al 1996) using primers 54 and 89c and was cloned between the SalI and SmaI sites of pUC18. pCT370 and pCT371: attR and attL of plasmids pMK24 and pMK25, respectively (Gottfried et al 2000), were cloned between ptac-k attR-t 1 t 2 -k attL Dorgai et al (1995) pLD205 ptac-attR-t 1 t 2 -attL Dorgai et al (1995) pLD300 attP-ptac-attB Dorgai et al (1995) pMK24 attR in pUC18 Gottfried et al (2000) pMK25 attL in pUC18 Gottfried et al (2000) pMK52 int in pCDNA3 Kolot et al (1999) pMK155 int-HK022 in pOK12 Malchin et al (2008) pMK171 int-k in pOK12 This work pMK221 pCMV-attP in pCDNA3 Kolot et al (2003) pMK223…”

Section: Cells Growth Conditions Plasmids and Primersmentioning

confidence: 99%

Arm site independence of coliphage HK022 integrase in human cells

et al. 2011

View full text Add to dashboard Cite

The integrase encoded by the lambdoid phage HK022 (Int-HK022) resembles its coliphage λ counterpart (Int-λ) in the roles of the cognate DNA arm binding sites and in controlling the direction of the reaction. We show here that within mammalian cells, Int-HK022 does not exhibit such a control. Rather, Int-HK022 recombined between all ten possible pairwise att site combinations, including attB × attB that was more effective than the conventional integrative attP × attB reaction. We further show that Int-HK022 depends on the accessory integration host factor (IHF) protein considerably less than Int-λ and exhibits stronger binding affinity to the att core. These differences explain why wild-type Int-HK022 is active in mammalian cells whereas Int-λ is active there only as an IHF-independent mutant.

show abstract

“…In all plasmids the "stop" site is a fragment that carries a transcription terminator that has been extracted from plasmid pBS302 (Sauer, 1993). attP originated from plasmid pKY110 (Kolot et al, 1996), attB from plasmid pNR185 (Nagaraja and Weisberg, 1990), attR from plasmid pMK24, and attL from plasmid pMK25 (Gottfried et al, 2000). In plasmids pMK218 and pMK189 the relevant fragments were cloned between the pCMV promoter and the GFP reading frame of plasmid pEGFP-N1 (ClonTech, Palo Alto, CA).…”

Section: Plasmid Constructionmentioning

confidence: 99%

Site‐specific recombination in human cells catalyzed by the wild‐type integrase protein of coliphage HK022

Kolot

Meroz

Yagil

2003

Biotech & Bioengineering

View full text Add to dashboard Cite

Abstract:The activity of the Integrase (Int) protein encoded by coliphage HK022 was tested in a human cell culture. Plasmids were constructed as substrates that carry the sites of the integration reaction (attP and attB) or the sites of excision (attL and attR). The site-specific recombination reactions were monitored in cis and in trans configurations by the expression of the green fluorescent protein (GFP) as a reporter. Cells cotransfected with the substrate plasmid(s) and with a plasmid that expresses the wild-type Int show efficient integration as well as excision in both configurations. The wild-type Int was active in the human cells without the need to supply the accessory proteins integration host factor (IHF) and excisionase (Xis) that are indispensable for the reaction in the bacterial host.

show abstract

A second site-specific recombination event in the lambdoid bacteriophage HK022

Cited by 5 publications

References 30 publications

Integrative recombination of Lactobacillus delbrueckii bacteriophage mv4: functional analysis of the reaction and structure of the attP site

Integrative recombination of Lactobacillus delbrueckii bacteriophage mv4: functional analysis of the reaction and structure of the attP site

Arm site independence of coliphage HK022 integrase in human cells

Site‐specific recombination in human cells catalyzed by the wild‐type integrase protein of coliphage HK022

Contact Info

Product

Resources

About