Lambda red recombineering in Escherichia coli occurs through a fully single-stranded intermediate.
The phage lambda-derived Red recombination system is a powerful tool for making targeted 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 Red mediates double-stranded DNA recombination remains uncertain. Current mechanisms posit a recombination intermediate in which both 5' ends of double-stranded DNA are recessed by λ exonuclease, leaving behind 3' overhangs. Here, we propose an alternative in which lambda 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-stranded gene insertion cassettes are recombinogenic and that these cassettes preferentially target the lagging strand during DNA replication. Furthermore, a double-stranded DNA cassette containing multiple internal mismatches shows strand-specific mutations cosegregating roughly 80% 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-targeting strand 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 by this work may facilitate further improvements to the versatility of lambda Red recombination.
Mosberg
J A
JA
Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA. jmosberg@fas.harvard.edu
Lajoie
M J
MJ
Church
G M
GM
D000483:Alleles; D010582:Bacteriophage lambda / Q000235:genetics*; D020137:Base Pair Mismatch / Q000235:genetics; D020090:Chromosome Segregation / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000235:genetics*; D004926:Escherichia coli / Q000821:virology*; D005818:Genetic Engineering / Q000379:methods*; D009154:Mutation / Q000235:genetics; D054735:Phosphorothioate Oligonucleotides / Q000235:genetics; D011995:Recombination, Genetic
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D054735:Phosphorothioate Oligonucleotides
Proc Natl Acad Sci U S A. 1997 Apr 1;94(7):2951-6
9096327
Mol Microbiol. 2008 Apr;68(1):66-74
18333884
J Mol Biol. 1987 May 5;195(1):75-87
2958632
Curr Protoc Mol Biol. 2007 Apr;Chapter 1:Unit 1.16
18265390
BMC Mol Biol. 2010 Jul 29;11:54
20670401
Nucleic Acids Res. 2005 Feb 24;33(4):e36
15731329
Science. 2006 May 19;312(5776):1044-6
16645050
Curr Protoc Mol Biol. 2009 Jan;Chapter 23:Unit 23.13
19170029
J Antimicrob Chemother. 2002 Mar;49(3):549-52
11864958
Genes Dev. 2000 Aug 1;14(15):1971-82
10921910
Plasmid. 2007 Sep;58(2):148-58
17434584
Proc Natl Acad Sci U S A. 2008 Feb 5;105(5):1626-31
18230724
Gene. 2000 Apr 4;246(1-2):321-30
10767554
Nat Genet. 2005 Mar;37(3):311-9
15731760
Nucleic Acids Res. 2000 Nov 15;28(22):E97
11071951
Proc Natl Acad Sci U S A. 2003 Jun 10;100(12):7207-12
12771385
Metab Eng. 2008 Jan;10(1):33-8
17959404
Nucleic Acids Res. 2003 Nov 15;31(22):6674-87
14602928
Mol Syst Biol. 2006;2:2006.0008
16738554
Annu Rev Genet. 2002;36:361-88
12429697
J Bacteriol. 1998 Apr;180(8):2063-71
9555887
Nano Lett. 2009 Dec;9(12):4302-5
19995086
Proc Natl Acad Sci U S A. 2000 May 23;97(11):5978-83
10811905
Genesis. 2001 Jan;29(1):14-21
11135458
Nucleic Acids Res. 2003 Mar 15;31(6):1585-96
12626699
Proc Natl Acad Sci U S A. 2003 Dec 23;100(26):15748-53
14673109
J Mol Biol. 2008 Dec 31;384(5):1098-105
18983848
Nat Biotechnol. 2000 Dec;18(12):1314-7
11101815
Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6640-5
10829079
Genomics. 2001 Apr 1;73(1):56-65
11352566
Protein Sci. 2010 May;19(5):967-73
20217896
Crit Rev Microbiol. 2009;35(3):197-220
19563302
Proc Natl Acad Sci U S A. 2001 Jun 5;98(12):6742-6
11381128
Nat Genet. 1998 Oct;20(2):123-8
9771703
Genetics. 1997 Nov;147(3):961-77
9383045
Methods Enzymol. 2007;421:171-99
17352923
Nat Protoc. 2009;4(2):206-23
19180090
Nature. 2009 Aug 13;460(7257):894-898
19633652