Abstract
The Red recombination system of bacteriophage lambda is widely used for genetic engineering because of its ability to promote recombination between bacterial chromosomes or plasmids and linear DNA species introduced by electroporation. The process is known to be intimately tied to replication, but the cellular functions which participate with Red in this process are largely unknown. Here two such functions are identified: the GrpE-DnaK-DnaJ chaperone system, and DNA polymerase I. Mutations in either function are found to decrease the efficiency of Red recombination. grpE and dnaJ mutations which greatly decrease Red recombination with electroporated DNA species have only small effects on Red-mediated transduction. This recombination event specificity suggests that the involvement of GrpE-DnaJ-DnaK is not simply an effect on Red structure or stability.
Highlights
The Red homologous recombination system of bacteriophage l operates via at least two distinct pathways [1]
In the Redsubstituted Escherichia coli strain, recombination between chromosomes brought together by the natural processes of conjugation or phage infection depends on double strand breaks, RecA, and RecFOR [5]
The sequences to be altered were replaced with the cat gene, with selection for chloramphenicol resistance; the cat gene was subsequently replaced by the altered sequence, with ampicillin or kanamycin enrichment for chloramphenicol-sensitive recombinants, as described previously [15]
Summary
The Red homologous recombination system of bacteriophage l operates via at least two distinct pathways [1]. The other, called the strand annealing pathway, is RecA-independent and replication-dependent These pathways were initially characterized by Stahl and coworkers in studies of recombination between phage chromosomes in infected cells (see [2] for a review). In the Redsubstituted Escherichia coli strain, recombination between chromosomes brought together by the natural processes of conjugation or phage infection depends on double strand breaks, RecA, and RecFOR [5]. In this respect, it resembles recombination between non-replicating l chromosomes in infected cells, in the latter case, RecFOR is not required because the phage encodes a protein, Orf, which can substitute [6] [7]
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