Checkpoint functions are required for normal S-phase progression in Saccharomyces cerevisiae RCAF- and CAF-I-defective mutants

Abstract

The chromatin-assembly factor I (CAF-I) and the replication-coupling assembly factor (RCAF) complexes function in chromatin assembly during DNA replication and repair and play a role in the maintenance of genome stability. Here, we have investigated their role in checkpoints and S-phase progression. FACS analysis of mutants lacking Asf1 or Cac1 as well as various checkpoint proteins indicated that normal rates of S-phase progression in asf1 mutants have a strong requirement for replication checkpoint proteins, whereas normal S-phase progression in cac1 mutants has only a weak requirement for either replication or DNA-damage checkpoint proteins. Furthermore, asf1 mutants had high levels of Ddc2.GFP foci that were further increased in asf1 dun1 double mutants consistent with a requirement for checkpoint proteins in S-phase progression in asf1 mutants, whereas cac1 mutants had much lower levels of Ddc2.GFP foci that were not increased by a dun1 mutation. Our data suggest that RCAF defects lead to unstable replication forks that are then stabilized by replication checkpoint proteins, whereas CAF-I defects likely cause different types of DNA damage.

Fig. 1.

asf1 mutants are not killed by either chronic or acute HU treatment. (A) Cells were plated on YPD and 50 mM HU plates and incubated at 30°C, as indicated. (B) Cell survival after acute treatment of cells with 200 mM HU for 2- or 4-h periods is shown as the percent of viable cells present before treatment.

Fig. 2.

asf1 caf-1 double mutants exhibit a higher proportion of cells in G₂/M and accumulate aberrant buds indicative of DNA damage. (A) Log-phase cells were stained with DAPI and analyzed by fluorescent microscopy. Each strain was scored for the percentage of cells with no buds, small buds, large buds, and aberrant buds. The distribution for CAF-I mutants, in the order stated above, were cac1 (18%, 48%, 30%, and 4%), cac2 (19%, 48%, 29%, and 3%), and cac3 (15%, 55%, 28%, and 2%). (B) The DNA content of the same series of mutants was determined by FACS analysis of cells from log-phase unsynchronized cultures.

Fig. 3.

asf1 and cac1 mutants require the function of different checkpoints for normal S-phase progression. FACS was used to monitor the rate of S-phase progression of various chromatin assembly/checkpoint double mutants after release from α-factor arrest. The proportion of cells in G₂/M was determined at the 40-min time point, at which at least 80% of wild-type cells have reached G₂/M. Error bars represent the SD. The percent of cells in G₂/M for the control strains was as follows: rad9, 81 ± 4%; rad24, 86 ± 6%; mec1 sml1, 85 ± 5%; rad53 sml1, 81 ± 4%; dun1, 80 ± 2%; chk1, 82 ± 10%; rfc5-1, 74 ± 7%; sgs1, 83 ± 7%.

Fig. 4.

Ddc2.GFP checkpoint protein complexes accumulate in asf1 and cac1 mutants. Live cells were analyzed in log phase by deconvolution microscopy to visualize Ddc2.GFP expression from an endogenous functional DDC2.GFP fusion gene resulting in Ddc2.GFP foci. (A) Cells are shown in Left (DIC), GFP fluorescence is shown in Center (FITC), and the two images are merged in Right (Merge). The experimental results quantified for asf1 (B) and cac1 (C) mutant cells are expressed as the percentage of cells containing Ddc2.GFP foci. The error bars presented are SDs. The control mutants rad9 DDC2.GFP and dun1 DDC2.GFP had 13% and 7% cells with Ddc2.GFP foci, respectively.

Fig. 5.

Levels of Rad53 phosphorylation in cac1 and asf1 cells compared with that in wild-type cells. A phosphopeptide (LLHS*NNTENVK; the asterisk indicates the phosphorylated Ser to its right, i.e., Ser-560) of Rad53 is used as an example. (A) The abundance of this phosphopeptide was unaffected by deletion of CAC1. (B) The abundance of this phosphopeptide increased to almost 5-fold in asf1 cells, compared with wild-type cells. In both cases, Rad53 peptides from wild-type cells were labeled by d0-leucine, whereas those from cac1 or asf1 cells were labeled by d10-leucine.

Fig. 6.

A model for the role of Asf1 in DNA replication and checkpoint activation. Asf1 plays an important role at replication forks during replication and repair. In the absence of Asf1, replication forks become unstable, leading to slower progression through S phase. Although somewhat unstable, these replication forks remain functional because of the presence of various replication checkpoint proteins, such as Ddc2, Rad53, and others, which play roles in replication forks stabilization. In the absence of a functional replication checkpoint, these somewhat unstable replication forks may collapse, thus leading to even slower S-phase progression, as well as conversion of the replication forks into structures that activate the DNA-damage checkpoint.