Assembly of RecA-like recombinases: distinct roles for mediator proteins in mitosis and meiosis

Abstract

Members of the RecA family of recombinases from bacteriophage T4, Escherichia coli, yeast, and higher eukaryotes function in recombination as higher-order oligomers assembled on tracts of single-strand DNA (ssDNA). Biochemical studies have shown that assembly of recombinase involves accessory factors. These studies have identified a class of proteins, called recombination mediator proteins, that act by promoting assembly of recombinase on ssDNA tracts that are bound by ssDNA-binding protein (ssb). In the absence of mediators, ssb inhibits recombination reactions by competing with recombinase for DNA-binding sites. Here we briefly review mediated recombinase assembly and present results of new in vivo experiments. Immuno-double-staining experiments in Saccharomyces cerevisiae suggest that Rad51, the eukaryotic recombinase, can assemble at or near sites containing ssb (replication protein A, RPA) during the response to DNA damage, consistent with a need for mediator activity. Correspondingly, mediator gene mutants display defects in Rad51 assembly after DNA damage and during meiosis, although the requirements for assembly are distinct in the two cases. In meiosis, both Rad52 and Rad55/57 are required, whereas either Rad52 or Rad55/57 is sufficient to promote assembly of Rad51 in irradiated mitotic cells. Rad52 promotes normal amounts of Rad51 assembly in the absence of Rad55 at 30 degrees C but not 20 degrees C, accounting for the cold sensitivity of rad55 null mutants. Finally, we show that assembly of Rad51 is induced by radiation during S phase but not during G(1), consistent with the role of Rad51 in repairing the spontaneous damage that occurs during DNA replication.

Figure 1

Generic model for assembly of recombinase on ssb-coated ssDNA. (A) Tracts of ssDNA form because of resection at DSB sites or stalling of polymerase. (B) ssb assembles into oligomeric filaments on tracts of ssDNA, removing secondary structures. (C) Mediator protein binds to ssb-coated DNA, causing a local remodeling of the ssb filament. (D) Recombinase initiates filament formation at sites of mediator-ssb-ssDNA. Recombinase filaments then elongate displacing ssb. (E) The elongated recombinase filament searches for homologous sequences. (F) Strand exchange occurs. The outgoing ssDNA strand is bound by ssb.

Figure 2

RPA assembles before Rad51 after γ-irradiation of G₂ arrested S. cerevisiae spheroplasts. A diploid yeast strain (NKY1314, SK-1 strain background) was grown in rich medium and arrested in G₂/M by treatment with nocodazole. Cells were spheroplasted by zymolyase treatment, irradiated to a dose of 50 krad with a ⁶⁰Co source, and resuspended in osmotically stabilized growth medium containing nocodazole. At the times indicated, culture aliquots were processed to obtain surface spread nuclei. Spread nuclei were indirectly immunostained with polyclonal guinea pig anti-Rad51(green) and rabbit anti-RPA70 (red), counter strained with a DNA-specific dye (DAPI), and examined by epifluorescence microscopy. The number of foci per nucleus was determined by visual examination of digital images. Details of the method have been published (29, 59). (A) Representative nuclei from the times indicated. Overlapping signals are yellow in these pseudocolored images. (B) Average number of foci detected per nucleus at the times indicated.

Figure 3

Role of S. cerevisiae mediators in assembly of Rad51 after γ-irradiation. Log phase diploid cells were irradiated to a dose of 100 krads and incubated in rich medium for the times indicated. All strains used were isogenic derivatives of SK-1. Mutants contain homozygous deletion alleles. Wilcoxon sum of ranks tests were used to assess the statistical significance of differences between samples referred to in the text. (A) The left column shows the distribution of focus counts at 0 and 2 h after irradiation from 20°C cultures, the right column from 30°C cultures. Focus counts from 50 unselected nuclei are displayed in ascending order. (B) Average number of foci plotted as a function of time for each of the strains examined. For detailed methods, see ref. .

Figure 4

Model for mediator-dependent coupling of ssDNA strand resection and recombinase assembly at DSBs during meiosis. (A) Strand-specific nuclease loads at DSB sites. (B) Mediators are loaded on DNA ends before nuclease proceeds a distance far enough to allow cytologically detectable amounts of RPA. (C) Rad51 is recruited to ends via mediator interactions, again before nuclease has proceeded far from the end. (D) Nucleolytic resection of DNA occurs in concert with elongation of the Rad51 filament. (E) Rad51 carries out homology search. (F) Strand exchange occurs with RPA loading on the outgoing ssDNA strand.

Figure 5

Radiation induction of Rad51 foci is cell cycle stage dependent. Subconfluent CHO cells were fractionated by centrifugal elutriation and aliquots of fractions assayed for DNA content via conventional methods (propidium iodide staining) on a Becton Dickinson FACScan. Raw data were analyzed to determine the percentage of the population in G₁, S, and G₂ by using cellquest software (Becton Dickinson). The remaining cells in each fraction were split into two fractions; one fraction was irradiated to a dose of 0.9 krad (9 Gy) x-rays with a Maxitron generator (General Electric), and the other served as an unirradiated control. All fractions were incubated in culture medium for 3 h, after which all fractions were analyzed a second time by FACS and also by immunostaining for Rad51 foci by using a previously published method (60). (A) Percentage of cells in G₁, S, and G₂ as estimated by FACS analysis of each of seven fractions obtained by elutriation. (B) FACS analysis of untreated fractions after 3-h incubation. (C) FACS analysis of irradiated fractions after 3-h incubation. (D) Percentage of Rad51-focus positive cells in each untreated fraction after a 3-h incubation in culture medium. (E) Percentage of Rad51-focus positive cells in each irradiated fraction after a 3-h incubation in culture medium.