Review
DNA recombination: the replication connection

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Abstract

Chromosomal double-strand breaks (DSBs) arise after exposure to ionizing radiation or enzymatic cleavage, but especially during the process of DNA replication itself. Homologous recombination plays a critical role in repair of such DSBs. There has been significant progress in our understanding of two processes that occur in DSB repair: gene conversion and recombination-dependent DNA replication. Recent evidence suggests that gene conversion and break-induced replication are related processes that both begin with the establishment of a replication fork in which both leading- and lagging-strand synthesis occur. There has also been much progress in characterization of the biochemical roles of recombination proteins that are highly conserved from yeast to humans.

Section snippets

The origins of double-strand breaks

In the laboratory, DSBs have classically been created by X-rays. More recently, repair of DSBs has been studied after their induction by endonucleases during such natural processes as meiosis or during programmed chromosome rearrangements such as V(D)J joining of mammalian immunoglobulin genes or switching of yeast mating-type genes. DSB repair after excision of transposable elements from a chromosome has also been analysed.

Although these repair events are important in the specialized cells

Break-induced replication

The first intimations of an origin- independent, recombination-dependent replication mechanism came from Mosig’s study of late DNA replication in phage T4 (Ref. 5), and our present understanding owes much to recent studies by many labs6, 7, 8 – especially those by the late Tokio Kogoma, who analysed recombination-dependent replication in Escherichia coli9. In this mechanism, one end of the DSB invades the template and establishes a replication fork (Fig. 1b) that could progress all the way to

Gene conversion

The DSB-repair model proposed by Szostak et al.16 comprises the following: invasion of a template by two 3′ single-stranded ends of the DSB; priming of new DNA synthesis from these 3′ ends; and formation of Holliday junctions, the four-stranded, branched structures whose resolution can yield both crossovers and noncrossovers (Fig. 2a). In this model, the newly synthesized DNA is semiconservative – that is, one new strand is present in the donor, and one is present in the recipient.

Such a

Repair-replication-fork capture

Increasingly, BIR and gene conversion seem to be closely related processes. Gene conversion, as in the case of BIR, can begin with the invasion of a donor template by one end of a DSB. Invasion establishes a replication fork, and both leading- and lagging-strand DNA synthesis occur. Gene conversion can result if the fork engages the second end of the DSB, which could anneal either to the displaced template strand in the migrating replication fork (Fig. 3a, Part 3) or to the newly synthesized

Genetic characterization of proteins involved in homologous recombination

Our understanding of the proteins involved in homologous recombinational repair of DSBs comes mostly from work in bacteria and bacteriophage, and work in yeast. In bacteria, the key recombination protein is RecA, which catalyses DNA strand exchange with a homologous duplex molecule25. In bacteria that lack RecA, nearly all recombination is eliminated.

Eukaryotes have one or more evolutionarily conserved homologs of RecA. One homolog, Rad51, is expressed in all cells; another homolog, Dmc1, is

Biochemical properties of proteins that catalyse homologous recombination

The most critical recombination protein, Rad52p, forms multimeric rings that can both bind to DNA ends and promote the annealing of complementary DNA strands37; human RAD52 has similar properties38. Rad52p associates with Rad51p38, 39, 40 and facilitates Rad51p-mediated strand exchange41, 42. Rad52p also interacts with the trimeric ssDNA-binding complex replication factor A (RPA)43. RPA is essential for DNA replication, but a separation-of-function mutation in the largest subunit, rfa1-t11,

Monitoring of recombination in vivo

Recently, cytological studies have examined the requirement for the formation of subnuclear complexes (foci) by Rad51p during meiosis in budding yeast. These foci are presumably sites of DNA-strand exchange. Deletion of rad52, rad55 or rad57 prevents formation of foci by Rad51p both in normal meiosis and after exposure to ionizing radiation50. This appears to be a very useful new approach to examining the early sequence of events that follows the appearance of DSBs.

Another valuable approach to

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