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Lambda Red Recombineering in Escherichia coli occurs through a fully single-stranded intermediate

Lambda Red Recombineering in Escherichia coli occurs through a fully single-stranded intermediate

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A Novel Mechanism for Red Recombination
A Novel Mechanism for Red Recombination

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Published by: fourin6 on Jan 10, 2013
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1
Lambda Red Recombineering in
 Escherichia coli
 occurs through a fully single-stranded intermediate
Mosberg, J. A.
*
 
§
, Lajoie, M. J.
*
 
§
, and Church, G. M.
 *
These authors contributed equally to this work.Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115,
§
Program in Chemical Biology, Harvard University, Cambridge, Massachusetts 02138
Genetics: Published Articles Ahead of Print, published on September 2, 2010 as 10.1534/genetics.110.120782
 
2Running Title: A Novel Mechanism for Red RecombinationKeywords: Recombineering, Lambda Red, Recombination, MechanismCorresponding Authors:Joshua A. Mosberg and Marc J. LajoieDepartment of GeneticsHarvard Medical School77 Avenue Louis Pasteur  New Research Building, Room 238Boston, MA 02115Phone: (617) 432-7670Fax: (617) 432-6513E-mails: jmosberg@fas.harvard.edu,mlajoie@fas.harvard.edu 
 
3ABSTRACTThe phage Lambda-derived Red recombination system is a powerful tool for makingtargeted genetic changes in
 Escherichia coli
, providing a simple and versatile method for generating insertion, deletion, and point mutations on chromosomal, plasmid, or BAC targets.However, despite the common use of this system, the detailed mechanism by which Lambda Redmediates double-stranded DNA recombination remains uncertain. Current mechanisms posit arecombination intermediate in which both 5′ ends of double-stranded DNA are recessed byLambda Exonuclease, leaving behind 3′ overhangs. Here, we propose an alternative in whichLambda Exonuclease entirely degrades one strand, while leaving the other strand intact as single-stranded DNA. This single-stranded intermediate then recombines via Beta recombinase-catalyzed annealing at the replication fork. We support this by showing that single-strandedgene insertion cassettes are recombinogenic, and that these cassettes preferentially target thelagging strand during DNA replication. Furthermore, a double-stranded DNA cassettecontaining multiple internal mismatches shows strand-specific mutations co-segregating roughly80% of the time. These observations are more consistent with our model than with previously proposed models. Finally, by using phosphorothioate linkages to protect the lagging-targetingstrand of a double-stranded DNA cassette, we illustrate how our new mechanistic knowledge can be used to enhance Lambda Red recombination frequency. The mechanistic insights revealed bythis work may facilitate further improvements to the versatility of Lambda Red recombination.INTRODUCTION

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