Resolving RAD51C function in late stages of homologous recombination

Abstract

DNA double strand breaks are efficiently repaired by homologous recombination. One of the last steps of this process is resolution of Holliday junctions that are formed at the sites of genetic exchange between homologous DNA. Although various resolvases with Holliday junctions processing activity have been identified in bacteriophages, bacteria and archaebacteria, eukaryotic resolvases have been elusive. Recent biochemical evidence has revealed that RAD51C and XRCC3, members of the RAD51-like protein family, are involved in Holliday junction resolution in mammalian cells. However, purified recombinant RAD51C and XRCC3 proteins have not shown any Holliday junction resolution activity. In addition, these proteins did not reveal the presence of a nuclease domain, which raises doubts about their ability to function as a resolvase. Furthermore, oocytes from infertile Rad51C mutant mice exhibit precocious separation of sister chromatids at metaphase II, a phenotype that reflects a defect in sister chromatid cohesion, not a lack of Holliday junction resolution. Here we discuss a model to explain how a Holliday junction resolution defect can lead to sister chromatid separation in mouse oocytes. We also describe other recent in vitro and in vivo evidence supporting a late role for RAD51C in homologous recombination in mammalian cells, which is likely to be resolution of the Holliday junction.

Figure 1

Schematic representation of the model showing repair of DSBs by homologous recombination. When a DSB occurs, it is processed to generate 3' single-stranded ends. RAD51 nucleoprotein microfilament is assembled on these ends, one of which invades the homologous DNA by strand displacement. This results in the formation of the D-loop. The invading strand primes DNA synthesis using the homologous DNA as a template. The second single-stranded DNA is also captured for priming DNA synthesis by using the displaced strand as a template. Extension of the 3' ends ultimately results in two cross-structures that hold the two homologs together, called Holliday junctions (HJs). Migration of HJs, called branch migration, results in the formation of heteroduplex regions. The double HJs are resolved by resolvases and, depending on the cleavage site, can either lead to crossover or non-crossover products. Resolution to yield crossover products requires symmetric cleavage of both HJs in opposite orientations.

Figure 2

A late role for RAD51C in meiotic recombination. RAD51C's late role in meiotic recombination is revealed in oocytes that were allowed to progress to metaphase II in vivo following hormonal treatment. A. At metaphase II, oocytes from control females show the presence of 20 pairs of chromatids, each consisting of two sister chromatids that are attached at their centromeres. B. Oocytes from infertile Rad51c mutant mice display a variety of chromosomal abnormalities. The majority of the mutant oocytes show precocious separation of sister chromatids. Inset in "D" is a higher magnification of the group of chromosomes showing an acentric chromatid (arrowhead) and a chromatid with two centromeres (double arrow). (Reproduced from The Journal of Cell Biology, 2007, 176:581–592, Copyright 2007, The Rockefeller University Press.)

Figure 3

Proposed connection between HJ resolution defect and abnormalities in Rad51c mutant oocytes at metaphase II. During meiosis, HJs are established between homologous chromosomes by the pachytene stage of prophase I using the homologous recombination machinery. At metaphase I, bivalents are pulled in opposite directions by microtubules attached to kinetochores of sister chromatids that are oriented toward the same pole. While centromeric cohesion is protected by Shugoshin to ensure that sister chromatids stay together during the reductional division, cohesion is released along the chromosome arms. During anaphase I, homologous chromosomes segregate to separate cells. In the absence of chiasmata, homologous chromosomes do not align properly at the metaphase plate, and this activates a spindle checkpoint resulting in metaphase I arrest. In Rad51c-deficient oocytes, meiosis progresses normally until anaphase I. However, due to accumulation of recombination intermediates, such as double Holliday junctions (dHJs), which hold the homologous chromosomes together, there is an increase in tension at the centromere due to the persistence of unresolved dHJs. The increased tension is thought to disrupt the sister chromatid cohesion at the centromere, resulting in the PSSC phenotype and fragmented chromosomes. Homologous chromosomes are shown in red and green; REC8 is shown in yellow; shugoshin is orange; and centromeres are shown in purple. (Reproduced from The Journal of Cell Biology, 2007, 176:581–592, Copyright 2007 The Rockefeller University Press.)