RecA-independent single-stranded DNA oligonucleotide-mediated mutagenesis

Abstract

The expression of Beta, the single-stranded annealing protein (SSAP) of bacteriophage lambda in Escherichia coli promotes high levels of oligonucleotide (oligo)-mediated mutagenesis and offers a quick way to create single or multiple base pair insertions, deletions, or substitutions in the bacterial chromosome. High rates of mutagenesis can be obtained by the use of mismatch repair (MMR)-resistant mismatches or MMR-deficient hosts, which allow for the isolation of unselected mutations. It has recently become clear that many bacteria can be mutagenized with oligos in the absence of any SSAP expression, albeit at a much lower frequency. Studies have shown that inactivation or inhibition of single-stranded DNA (ssDNA) exonucleases in vivo increases the rate of SSAP-independent oligo-mediated mutagenesis. These results suggest that lambda Beta, in addition to its role in annealing the oligo to ssDNA regions of the replication fork, promotes high rates of oligo-mediated mutagenesis by protecting the oligo from destruction by host ssDNA exonucleases.

Figure 1.. Replisome invasion model of single-stranded DNA oligo-mediated mutagenesis

Top: following electroporation into Escherichia coli host cells expressing bet, the ssDNA oligo (red arrow) is bound by the ring-shaped oligomer of the λ Beta protein (blue). The 3′ end of the primer is designated by the arrowhead. Beta promotes annealing of the oligo to single-stranded regions of the DNA replication fork. The green cylinder represents the replicative helicase DnaB; for clarity, the rest of the replisome is not drawn. The oligo can be complementary to either the lagging strand (a) or leading strand (b) templates. (a) Once annealed to the lagging strand template, the Beta ring is removed, perhaps by some interaction with a component of the replication fork. The oligomer is extended by DNA polymerase I and incorporated into the chromosome by ligation to an adjacent Okazaki fragment. (b) Beta promotes annealing of the oligo just ahead of the polymerase on the leading strand template. After annealing, the Beta ring is removed, perhaps by some interaction with a component of the replication fork. The leading strand polymerase then bumps into the 5′ end of the oligo, leaves its template, and re-initiates DNA synthesis downstream using the 3′ end of the oligo as a primer. Such jumps in leading strand polymerases are thought possible given the gaps in leading strand synthesis that are observed in in vivo studies [33].