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Arm site independence of coliphage HK022 integrase in human cells

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Abstract

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.

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Abbreviations

ApR :

Ampicillin resistance

att :

Attachment site

bp:

Base pairs

CMV:

Cytomegalovirus

FACS:

Fluorescent-activated cell sorting

FIS:

Factor for inversion specificity

GFP:

Green fluorescent protein

HygR :

Hygromycin resistance

IHF:

Integration host factor

Int:

Integrase

KmR :

Kanamycin resistance

LB:

Luria–Bertani broth

MW:

Molecular weight markers

NeoR :

Neomycin resistance

wt:

Wild type

X-gal:

5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside

Xis:

Excisionase

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Acknowledgments

N.M. is supported by the Leon Reich Scholarship Fund. Gabi Kaufman assisted us with science and language. László Dorgai sent us plasmid pAG159. Research was supported by grant No. MB-8713-08 from BARD, the United States-Israel Binational Agricultural Research and Development Fund and grant No. 1062/2008 from G.I.F., the German Israeli Foundation for Scientific Research and Development to E.Y. and M.K.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Tel-Aviv University, 69978, Tel-Aviv, Israel

    Natalia Malchin, Chen N. Tuby, Ezra Yagil & Mikhail Kolot

Authors
  1. Natalia Malchin
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  2. Chen N. Tuby
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  3. Ezra Yagil
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  4. Mikhail Kolot
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Correspondence to Mikhail Kolot.

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Communicated by D. Andersson.

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Malchin, N., Tuby, C.N., Yagil, E. et al. Arm site independence of coliphage HK022 integrase in human cells. Mol Genet Genomics 285, 403–413 (2011). https://doi.org/10.1007/s00438-011-0614-3

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  • DOI: https://doi.org/10.1007/s00438-011-0614-3

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