Illegitimate recombination includes those events that do not fall into any of the more clearly defined categories of recombination (homologous recombination, site-specific recombination, and transposition). Illegitimate recombination may itself include an assortment of types of events. Parental DNA molecules or DNA segments in illegitimate recombination often share a few homologous base pairs. Some of these events result from DNA replication errors and others from joining broken DNA ends. Illegitimate recombination generates deletions and translocations and therefore can have profound genetic consequences.
One type of illegitimate recombination widely studied in bacteria results from slippage between the template and newly replicated DNA strands. The prototype for this mechanism is that suggested by Streisenger et al. (1) for generating frameshift mutations at points of repeated nucleotides. For example, DNA containing on one strand A5 can generate A4 or A6 by loss or gain of a base pair by the following proposed mechanism. Replication pauses within the repeat. The newly synthesized strand unwinds and anneals with the template, but out of register, leaving one or more nucleotides as a single-stranded loop on either the template or the newly synthesized strand. Replication resumes, and this newly synthesized strand has fewer or more nucleotides than the parent. In the absence of repair synthesis, another round of faithful replication fixes the mutation.
A similar event can occur between direct repeats of any sequence that may or may not be separated by intervening base pairs. If the repeats do not themselves have internal repeats, interaction between them produces a deletion or duplication of one copy of the repeat plus the intervening sequence. One could imagine that these events occur by homologous recombination where the repeats are out of register (unequal crossing over), but in Escherichia coli these events are independent of known homologous recombination functions. The events are not reciprocal, that is, a deletion is not formed concurrently with a duplication. Deletions can occur between repeats separated by thousands of base pairs, although the frequency decreases as this separation becomes larger (2).
Linear DNA introduced into mammalian cells by transfection often undergoes illegitimate recombination by integration into the genome or by end-joining. End-joining has been extensively studied and occurs by ligation of the ends without any apparent requirement for homology. If excision of nucleotides from one or both ends occurs before or after transfection, a few homologous base pairs may be exposed, and annealing may occur between them. Filling of gaps and ligation can generate a novel joint. These events may be paradigms for integration of exogenous linear DNA and for chromosomal translocations. These events typically occur between DNA molecules sharing little or no homology and may occur between broken chromosome ends that swap partners and rejoin.
The DNA ends that provoke illegitimate recombination may be generated by a variety of mechanisms. Linear DNA may be introduced into cells, as in transfection. Restriction Enzymes may be induced to cut DNA at special sites in cells. DNA topoisomerases make transient double-strand breaks that may be illegitimately rejoined. This process is stimulated by topoisomerase inhibitors. Pausing or termination of DNA replication generates single-strand gaps whose ends may provoke illegitimate recombination, perhaps after conversion of the gap into a double-strand break. The variety of mechanisms that generate breaks may account for the variety of types of illegitimate recombination observed in cells.
Illegitimate recombination is not entirely a haphazard process. Regulated forms of this process occur in generating functional immunoglobulin genes by gene rearrangements and in eliminating micronuclear DNA in Tetrahymena macronucleus development. Other types of recombination previously considered illegitimate are recognized now as site-specific recombination and transposition. Further studies of illegitimate recombination may reveal unappreciated mechanisms and the biological roles of these events.
