In the Lac assay system in E. coli 
, amplification of the lac
operon to 20–100 copies occurs in response to the stress of starvation 
. The novel junctions of the amplified segments (amplicons) show that endpoints occurred at sites of microhomology of 2–15 bp 
. Some of the amplicons are complex, containing both direct and inverted repeats. Many others cannot be identified by outward-facing polymerase chain reaction (an observation also encountered frequently for PLP1
duplication junction analysis 
), which would reveal the junctions of simple tandem repeats, and so are presumed to be complex, rather than simple tandem repeats 
. By these criteria, about 25% of amplicons are complex. Thus, with respect to microhomology and complexity, the chromosomal structural changes in this system resemble those found in nonrecurrent events in human genomic disorders.
Homologous recombination requires RecA protein (Rad51 in eukaryotes) (reviewed in 
). Microhomology-mediated deletion formation in E. coli
(less than 25 nucleotides of homology) has long been known to be RecA-independent 
. RecA-independent short homology-mediated deletions (25–50 nucleotides) have previously been attributed to template switching within a replication fork during DNA replication (reviewed in 
). The evidence for this is, first, that mutations in genes encoding replication functions affect the formation of these events; second, that mutations affecting post-replicational mismatch repair affect them, placing the event very near to the replication fork; third, that mutation of 3′ exonucleases has an effect that is consistent with the ends being used to prime DNA synthesis; and fourth, that it is very difficult to obtain mutations affecting the process by transposon mutagenesis, suggesting essential functions.
In the E. coli
Lac system, study of genetic requirements of stress-induced amplification has revealed some details of the mechanism. First, the events involve 3′ DNA ends. This is seen by an increase in amplification when a 3′ exonuclease gene (xonA
) is deleted, and a decrease when the 3′ exonuclease is over-expressed. Similar manipulation of 5′-exonuclease has no effect 
. This suggests that amplification results from free 3′ ends in the cell most of which are normally removed by exonuclease. As above, the involvement of 3′ ends but not 5′ ends is consistent with priming of DNA synthesis.
Second, lagging-strand processing at replication forks is implicated by a requirement for the 5′ exonuclease domain of DNA polymerase I (Pol I) 
. Pol I is involved in lagging-strand replication, base excision repair, and nucleotide excision repair, but these excision repair processes are not involved in amplification 
, so lagging strands at replication forks are implicated in amplification.
Third, there is a requirement for the proteins of double-strand break (DSB) repair by homologous recombination 
(the RecBC system, reviewed in 
). That this is actually a requirement for DSB repair (not just the proteins) is shown by the discovery that in vivo double-strand cleavage of DNA near lac
enhances amplification rates 
Taken together, these observations suggest a model for amplification in the Lac system in E. coli
in which replication is restarted at sites of repair of DNA double-strand ends 
. The hypothesis proposed was that template switching occurs during replication restart at stalled replication forks. Because the distances involved exceed the lengths that are expected to be exposed as single-stranded at a single replication fork, it was proposed that the switches occurred between different replication forks 
The idea that chromosomal structural changes originate from DNA replication has received support from a study of microhomology-mediated SD formation in yeast 
. These authors support the idea that the mechanism of SD formation involves replication by showing that its frequency is enhanced by treatment with camptothecin and is dependent on Pol32, a component of Polδ (discussed below). Camptothecin is a topoisomerase I inhibitor that leaves nicks in DNA. These nicks are believed to become collapsed forks when a replication fork reaches them. Thus, increasing the frequency of fork collapse increases the frequency of duplication formation. These authors also report that situations that lead to fork stalling rather than collapse have little effect on the frequency of duplication formation 
. Thus, it appears that the substrate for duplication is a single double-strand end at a collapsed replication fork.
This long-distance template-switch model was also used by Lee et al. 
to explain the observations of nonrecurrent chromosomal changes seen in Pelizaeus-Merzbacher disease discussed above and the juxtaposition of multiple genomic sequences normally separated by large genomic distances 
. Experiments on the integration of nonhomologous DNA into mammalian cells revealed microhomology junctions and insertion of sequence from other parts of the genome at the junctions. These observations were interpreted in terms of a similar model of repeated copying and switching to another template