18 2 Trackable Multiplex Recombineering (TRMR) and Next-Generation Genome Design Technologies
and, importantly, all along the genome during chromosome replication. Studies
show that the ssDNA substrate anneals to the chromosome in a strand-biased
manner, which correlates with the direction of DNA replication [19, 20]. These
results suggest that the ssDNA annealing is directed to the lagging strand of the
replication fork via its homology regions, where it essentially acts as an exoge-
nous DNA-based Okazaki fragment [16, 21]. Overall, the recombineering
Leading strand Lagging strand
Okazaki
fragments
DNA to be
incorporated
Homology arms
Replication fork
5 ′
5 ′ 5 ′
3 ′
3 ′ 3 ′
Exo
dsDNA ssDNA
Beta
RecBCD
Gam
Figure 2.1 The λ-Red system and the replication fork annealing model of recombination.
Either a double- or single-stranded recombineering substrate, consisting of the DNA
sequence to be inserted flanked by homology arms, is transformed into cells. The λ-Red
proteins facilitate recombination by digesting one strand of DNA in the case of dsDNA (Exo),
by inhibiting RecBCD nuclease activity (Gam), and by protecting and conveying the ssDNA to
the replication fork (Beta). Then, the ssDNA acts as a mismatched Okazaki fragment and binds
to the lagging strand via its homology arms. This process results, upon completion of cell
duplication, with one wild-type daughter cell and one recombineered, heterozygous-like
daughter cell. Reprinted with permission from Pines et al. 2015 [15]. Copyright 2015 American
Chemical Society.