Rad52 recruitment is DNA replication independent and regulated by Cdc28 and the Mec1 kinase

Abstract

Recruitment of the homologous recombination machinery to sites of double-strand breaks is a cell cycle-regulated event requiring entry into S phase and CDK1 activity. Here, we demonstrate that the central recombination protein, Rad52, forms foci independent of DNA replication, and its recruitment requires B-type cyclin/CDK1 activity. Induction of the intra-S-phase checkpoint by hydroxyurea (HU) inhibits Rad52 focus formation in response to ionizing radiation. This inhibition is dependent upon Mec1/Tel1 kinase activity, as HU-treated cells form Rad52 foci in the presence of the PI3 kinase inhibitor caffeine. These Rad52 foci colocalize with foci formed by the replication clamp PCNA. These results indicate that Mec1 activity inhibits the recruitment of Rad52 to both sites of DNA damage and stalled replication forks during the intra-S-phase checkpoint. We propose that B-type cyclins promote the recruitment of Rad52 to sites of DNA damage, whereas Mec1 inhibits spurious recombination at stalled replication forks.

Figure 1

Rad52 foci do not form in the absence of Cdc28–B-type cyclin activity. (A) cdc4-1 cells are first arrested by shifting to non-permissive temperature for 2 h. cdc4-1 cells do not form Rad52 foci in response to 40 Gy IR, in contrast to WT cells. (B) FACS profile of DNA content of WT and cdc4-1 cells upon shift to 37°C upon exposure to IR. (C) Cells containing an analogue-sensitive allele of Cdc28, cdc28-as1, are exposed to 40 Gy IR in the presence and absence of the specific inhibitor 1-NMPP1.

Figure 2

Rad52 foci form in the absence of bulk DNA replication. (A) cdc6-1 cells are first held at the non-permissive temperature for 120 min, exposed to 40 Gy IR, and samples are taken for microscopy. cdc6-1 mutant cells show elevated levels of spontaneous Rad52 foci (time 0), and also form foci in response to IR. (B) FACS analysis showing DNA content of the cells shown in (A). (C) cdc7-4 cells are first arrested in G1 with α-factor, then shifted to the non-permissive temperature for 2 h. Cells were then exposed to 40 Gy IR while in α-factor arrest and released 30 min after exposure. (D) Representative FACS samples of cdc7-4 cells shown in (C).

Figure 3

HU suppresses Rad52 but not Mre11 or Rfa1 focus formation. (A) Mre11 foci form in response to IR in HU-arrested cells. (B) Rfa1 foci form in response to IR during HU arrest. White arrowheads indicate bright IR-induced foci, whereas red arrowheads point to fainter foci characteristic of those observed during DNA replication. (C) Rad52 focus formation in HU-treated cells over time. HU-arrested cells do not form appreciable levels of Rad52 foci in response to 40 Gy IR in comparison to untreated cells. (D) Rad52 focus formation in HU-treated cells at high doses of IR. HU-arrested cells are exposed to a range of IR doses, and then assayed for Rad52 focus formation after 60 min. WT budded cells form Rad52 foci at 40 Gy IR, whereas unbudded G1 cells form Rad52 foci at IR doses of 400 Gy and higher. HU-arrested cells, on the other hand, do not form Rad52 foci at any level of IR dose tested. (E) HU does not inhibit Rad52 focus formation in G2-arrested cells. Cells first arrested in G2/M with nocodazole are treated with 100 mM HU. Rad52 forms foci in response to IR in G2/M-arrested cells, even in the presence of HU. (F) The addition of HU does not dissociate Rad52 foci formed in response to IR. Cells first exposed to IR are treated with HU 90 min after exposure. The addition of HU does not lead to the disappearance of the Rad52 foci that form in response to IR.

Figure 4

Caffeine inhibits the Rad53-mediated DNA damage checkpoint response. (A) Caffeine inhibits phosphorylation of Rad53 in response to 40 Gy of IR. Asynchronously growing cells are incubated in 20 mM caffeine, and half the culture is then exposed to IR. Extracts were taken from cells and blotted for Rad53 (filled-in arrowhead). Cells exposed to 40 Gy IR exhibit Rad53 hyperphosphorylation (open arrowhead), whereas cells treated with caffeine do not. (B) Caffeine-induced inhibition of Mec1/Tel1 kinase activity blocks damage-induced, but not S phase, degradation of Sml1. The levels of Sml1 protein in vivo were assessed by monitoring YFP–Sml1 fluorescent protein levels. WT cells normally have high levels of cytoplasmic Sml1 and rapidly degrade Sml1 in response to exposure to IR. S-phase cells (arrowheads) degrade Sml1 in an Mec1-independent manner. Caffeine-treated cells degrade Sml1 in S-phase cells (arrowheads), similar to WT, but do not degrade Sml1 in response to damage, indicating that the IR-induced damage requires Mec1/Tel1 activity, whereas the S-phase degradation does not. (C) HU treatment induces Rad53 phosphorylation; however, hyperphosphorylated Rad53 (open arrowhead) does not disappear with the addition of caffeine to HU-treated cells but does abrogate cell cycle arrest (FACS analysis, data not shown), indicating that persistent Mec1 activity is required for maintenance of the intra-S checkpoint.

Figure 5

Rad52 recruitment during S phase. (A) Rad52 focus formation after caffeine treatment. The addition of 20 mM caffeine to asynchronously growing cells induces a small increase in Rad52 focus formation by 120 min, yet this increase is not statistically significant, suggesting that the Rad52 foci observed in mec1Δ cells accumulate over multiple cell divisions in response to spontaneous DNA damage. (B) Caffeine treatment causes increased Rad52 foci in G1 cells in response to IR. The addition of caffeine does not change Rad52 focus formation in response to IR in budded cells. However, the foci in the unbudded cell population increases, consistent with loss of the Mec1-dependent G2/M checkpoint. (C) HU-arrested cells treated with caffeine form Rad52 foci in response to IR. Cells are first treated with 100 mM HU, then with 20 mM caffeine for 30 min, and finally exposed to IR. (D) Rad52 foci colocalize with PCNA in HU-arrested cells treated with caffeine. Cells treated first with 100 mM HU for 90 min and then with 20 mM caffeine for 30 min form Rad52–YFP foci and these foci colocalize with CFP–Pol30 foci present in S-phase cells. (E) Model for Rad52 recruitment to damage during S phase. After DNA damage, Mec1/Rad53 activity prevents replication fork collapse at lesions by stabilizing stalled replication forks, channelling lesion bypass through BER, NER, and so on. In the absence of Mec1/Rad53 activity, replication forks collapse, requiring recombination for replication restart, manifested as increased Rad52 foci.