19. Summary

14.19 Summary

Recombination involves the physical exchange of parts between corresponding DNA molecules. This results in a duplex DNA in which two regions of opposite parental origins are connected by a stretch of hybrid (heteroduplex) DNA in which one strand is derived from each parent. Correction events may occur at sites that are mismatched within the hybrid DNA. Hybrid DNA can also be formed without recombination occurring between markers on either side. Gene conversion occurs when an extensive region of hybrid DNA forms during normal recombination (or between nonallelic genes in an aberrant event) and is corrected to the sequence of only one parental strand; then one gene takes on the sequence of the other.

Recombination is initiated by a double-strand break in DNA. The break is enlarged to a gap with a single-stranded end; then the free single-stranded end forms a heteroduplex with the allelic sequence. The DNA in which the break occurs actually incorporates the sequence of the chromosome that it invades, so the initiating DNA is called the recipient. Hot spots for recombination are sites where double strand breaks are initiated. A gradient of gene conversion is determined by the likelihood that a sequence near the free end will be converted to a single strand; this decreases with distance from the break.

The recombination event is not well understood at the level of the chromosome, but the properties of yeast mutants, and the relative timing of events during meiosis, suggest that the synaptonemal complex may be the consequence of, rather than a prerequisite for, the initiation of the recombination event.

The only enzymes whose activities have been characterized in recombination are coded by the rec and ruv loci of E. coli. RecA has the ability to synapse homologous DNA molecules by sponsoring a reaction in which a single strand from one molecule invades a duplex of the other molecule. Heteroduplex DNA is formed by displacing one of the original strands of the duplex. The RecBCD nuclease binds to DNA on one side of a chi sequence, and then moves to the chi sequence, unwinding DNA as it progresses. A single-strand break is made at the chi sequence. chi sequences provide hotspots for recombination. RuvA and RuvB act at a heteroduplex, and RuvC cleaves Holliday junctions.

Recombination, like replication and (probably) transcription, requires topological manipulation of DNA. Topoisomerases may relax (or introduce) supercoils in DNA, and are required to disentangle DNA molecules that have become catenated by recombination or by replication. The enzymes involved in site-specific recombination have actions related to those of topoisomerases. Phage lambda integration requires the phage Int protein and host IHF protein and involves a precise breakage and reunion in the absence of any synthesis of DNA. The reaction involves wrapping of the attP sequence of phage DNA into the nucleoprotein structure of the intasome, which contains several copies of Int and IHF; then the host attB sequence is bound, and recombination occurs. Reaction in the reverse direction requires the phage protein Xis.

Bacteria contain systems that maintain the integrity of their DNA sequences in the face of damage or errors of replication and that distinguish the DNA from sequences of a foreign source.

Repair systems can recognize mispaired, altered, or missing bases in DNA, or other structural distortions of the double helix. Excision repair systems cleave DNA near a site of damage, remove one strand, and synthesize a new sequence to replace the excised material. Three excision repair systems in E. coli can be distinguished by the lengths of the regions that are excised. The dam system is involved in correcting mismatches generated by incorporation of incorrect bases during replication, and the uvr genes are involved in both of the other systems for general repair. Recombination Vrepair systems retrieve information from a DNA duplex and use it to repair a sequence that has been damaged on both strands. The recBC and recF pathways identify two different systems. The recA product may be involved in repair pathways in one of its capacities, the ability to synapse molecules of DNA.

The other capacity of recA is the ability to induce the SOS response. RecA is activated by damaged DNA in an unknown manner. It triggers cleavage of the LexA repressor protein, thus releasing repression of many loci, and inducing synthesis of the enzymes of both excision repair and recombination-repair pathways. Genes under LexA control possess an operator SOS box. RecA also directly activates some repair activities. Cleavage of repressors of lysogenic phages may induce the phages to enter the lytic cycle.