An Introduction to
Break-induced Recombination

A number of models have been put forth over the years to explain how DNA recombination occurs. The basis of these models lies in what has been learned of recombination from yeast and other fungi. Fungi have been studied intensively because they possess certain properties such as the ability to yield four viable meiotic products (spores) that can be assayed and analyzed. For example, a mutation that is heterozygous will give rise to two mutant spores and two wild type spores, a 2+:2- pattern that is considered to be classically Mendelian. The appearance of non-Mendelian patterns such as 1+:3- (a "gene conversion") or the presence of "sectored" colonies (colonies with a divided phenotype) gave rise to a series of models to explain these events. While it is interesting how different models evolved, we concentrate here on a basic working model of DSB-induced recombination.

The diagram below shows a working model of how DNA double strand break induced recombination can occur.

Strand Resection. One strand of the duplex is degraded by a nuclease starting from the DSB.
Strand Invasion. The single stranded tail searches for a homologous sequence elsewhere in the genome. If it finds a sequence it will invade the duplex and form a heteroduplex DNA.
Annealing and Synthesis. Strand synthesis initiates from the 3' ends. The other single-stranded tail can anneal to the displaced strand.
Holliday Junction. DNA synthesis and strand displacement leads to formation of a second crossover structure called a Holliday Junction.
Resolution of Holliday junctions and Crossing over. Cleavage of the Holliday junctions can occur via nucleases in two ways. Cleavage of both junctions along different axes will lead to a crossover of the distal arms. On the other hand, cleavage along the same axes will leave the distal arms unchanged.
In this model gene conversion can be described as the transfer of genetic information from one DNA strand to a recipient sequence. For example, if the invading strand contains a mutation, then that mutation can be transferred to the recipient duplex DNA. The heteroduplex would contain mismatched DNA that could be repaired to either the mutation or the wild type sequence. If mismatch is changed to the mutated sequence then the wild type sequence was gene converted to the mutated sequence (see diagram). Sectored colonies result when the mismatch is not corrected and the two strands segregate to different cells following DNA replication and mitosis.
Mismatch is corrected to wild type or to the mutant allele.
Uncorrected sequences segregate into different cells at mitosis and form a sectored spore colony.

 More generally, a gene conversion refers to the unidirectional transfer of genetic information as opposed to a reciprocal exchange of information. In S. cerevisiae, gene conversion is recognized as a fundamental recombination process that can occur with or without crossing over of adjacent sequences.

Meiotic recombination
Experimental evidence for this model comes in the form of genetic and physical evidence. Physical analysis of DNA isolated from cells undergoing meiosis, for example, shows that DSBs are formed and they occur at recombination hotspots. For example, they occur in regions of high gene conversion that decreases with increasing distance from the DSB, a phenomenon termed polarity. Furthermore, these experiments also showed that DSBs lead to the formation of single stranded tails. Gel electrophoretic experiments have also provided evidence for structures that appear to be double Holliday structures. Gene conversion gradients are lost in mutants deficient in mismatch repair, indicating that gene conversion tracts are formed by long stretches of heteroduplex DNA. Special mutations that cannot be mismatch-corrected also indicate the presence of long heteroduplex tracts.

Mitotic recombination.

Break-induced recombination can also be studied in cells growing mitotically. In our lab we study this by inducing the HO endonuclease which initiates the process of mating type switching in yeast. The HO endonuclease cleaves the DNA sequence at MAT, the mating type locus, that determines the cell's mating type (either a or alpha). The DSB causes a gene conversion event to occur using either of two donor sequences, called HML and HMR, leading to the unidirectional transfer of mating type information from HML or HMR to MAT. Which donor is preferred depends on a sequence called the recombination enhancer which we consider in another part of this web site (Link to donor preference).

 To study DSB-induced recombination we have placed the HO gene under the control of a galactose promoter and moved the HO endonuclease recognition sequence from its normal position at the MAT locus and placed it in other contexts. As an example of gene conversion, the HO cut site (HOcs) was placed within one copy of the lacZ sequence on a plasmid containing a second copy of lacZ. The DSB created by HO endonuclease can be repaired using the donor copy of lacZ. The HOcs is lost during this process due to the unidirectional nature of gene conversion.
Evidence for several other mechanisms of DSB-repair, such as Synthesis Dependent Strand Annealing (SDSA), Single Strand Annealing (SSA), Break induced Replication (BIR) and Non-Homologous End Joining (NHEJ). We encourage readers to explore other sections of this web site describing these processes.
*We thank Monica Colaiacovo for letting us use her drawing on this page.