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. 2015 Apr;199(4):1047-63.
doi: 10.1534/genetics.114.173856. Epub 2015 Feb 18.

Replisome function during replicative stress is modulated by histone h3 lysine 56 acetylation through Ctf4

Pierre Luciano  1 , Pierre-Marie Dehé  1 , Stéphane Audebert  1 , Vincent Géli  1 , Yves Corda  2
Affiliations

Replisome function during replicative stress is modulated by histone h3 lysine 56 acetylation through Ctf4

Pierre Luciano et al. Genetics. 2015 Apr.

Abstract

Histone H3 lysine 56 acetylation in Saccharomyces cerevisiae is required for the maintenance of genome stability under normal conditions and upon DNA replication stress. Here we show that in the absence of H3 lysine 56 acetylation replisome components become deleterious when replication forks collapse at natural replication block sites. This lethality is not a direct consequence of chromatin assembly defects during replication fork progression. Rather, our genetic analyses suggest that in the presence of replicative stress H3 lysine 56 acetylation uncouples the Cdc45-Mcm2-7-GINS DNA helicase complex and DNA polymerases through the replisome component Ctf4. In addition, we discovered that the N-terminal domain of Ctf4, necessary for the interaction of Ctf4 with Mms22, an adaptor protein of the Rtt101-Mms1 E3 ubiquitin ligase, is required for the function of the H3 lysine 56 acetylation pathway, suggesting that replicative stress promotes the interaction between Ctf4 and Mms22. Taken together, our results indicate that Ctf4 is an essential member of the H3 lysine 56 acetylation pathway and provide novel mechanistic insights into understanding the role of H3 lysine 56 acetylation in maintaining genome stability upon replication stress.

Keywords: Ctf4; H3K56 acetylation; Mms22; replicative stress; replisome.

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Figures

Figure 1
Figure 1
Asf1 interaction with histone H3 is crucial for viability of yeast rrm3∆ cells. (A) Deletion of ASF1 is lethal in rrm3∆ cells. Tetrad dissection of the diploid strain asf1∆/ASF1 rrm3∆/RRM3. In this and subsequent figures, the four spores from a given tetrad are in a vertical line on a YPD plate. Four representative tetrads are shown after 3 days (left) and after 5 days (right) at 30°. Squares indicate the rrm3∆ single mutants. Circles indicate the asf1∆ single mutants. Assuming that 2:2 segregation of the marker allows one to identify asf1∆ rrm3∆ double mutants (indicated by dashed circles). (B) Wild-type and asf1∆ rrm3∆ cells (from microcolonies shown in A, right) analyzed by differential interference contrast and 4′,6-diamidino-2-phenylindole staining. Almost all asf1∆ rrm3∆ cells analyzed have no distinct nucleus compared to wild-type cells. (C) Effects of Asf1 interactions on the viability of rrm3∆ cells. Tetrad analysis of the meiotic progeny of asf1∆/ASF1 rrm3∆/RRM3 diploid cells expressing asf1-D37R E39R (i) or asf1-V94R (ii) mutated forms of ASF1 from plasmid pRS314. The presence of asf1∆ rrm3∆ spores is indicated by dashed circles. asf1∆ rrm3∆ spores expressing asf1-D37R E39R (i) or asf1-V94R (ii) are indicated by circles. A plus sign (+) indicates spores carrying the plasmid. wt, A1, R3, and A1R3 indicate wild-type, asf1∆, rrm3∆, and asf1∆ rrm3∆ spores, respectively. (iii) Tetrad analysis of the meiotic progeny of rad53-ALRR/RAD53 rrm3∆/RRM3 diploid cells. Circles indicate the rad53-ALRR mutant. Dashed circles indicate the rad53-ALRR rrm3∆ double mutant. wt, R3, ALRR, and ALRR R3 indicate wild-type, rrm3∆, rad53-ALRR, and rad53-ALRR rrm3∆ spores, respectively.
Figure 2
Figure 2
Hyperacetylation and hypoacetylation of lysine 56 of histone H3 affect rrm3∆ cells differently. (A) H3K56R mutation is lethal in rrm3∆ cells. Tetrads from diploids for hht1∆-hhf1∆/HHT1-HHF1 hht2∆-hhf2∆/HHT2-HHF2 rrm3∆/RRM3 expressing HHF1 and hht1-K56R from a centromeric plasmid were dissected and analyzed for the presence of auxotrophic markers. Dashed circle indicates rrm3∆ spore expressing H3K56R as sole source of histone H3. (B) rrm3∆ cells are viable with constitutively acetylated H3K56. The hst3∆/HST3 hst4∆/HST4 rrm3∆/RRM3 sir2∆/SIR2 diploid strain was dissected. The presence of rrm3∆ hst3∆ hst4∆ and rrm3∆ sir2∆ hst3∆ hst4∆ mutants is indicated by a circle and by dashed circles, respectively.
Figure 3
Figure 3
A Cac1/Rtt106-independent function of H3K56ac is required for viability of rrm3∆ cells. (A) Defective Cac1/Rtt106-dependent chromatin assembly does not cause lethality in the absence of RRM3. Tetrads from the rtt106∆/RTT106 cac1∆/CAC1 asf1∆/ASF1 rrm3∆/RRM3 diploid strain were dissected. Diamonds indicate cac1∆ rtt106∆ mutants. Hexagon indicates the rtt106∆ rrm3∆ mutant. Square indicates the cac1∆ rrm3∆ mutant. Circle indicates the cac1∆ rtt106∆ rrm3∆ mutant. Dashed circles indicate asf1∆ rrm3∆ mutants. Triangle indicates asf1∆ cac1∆ mutant. (B) Effects of cac1∆ and rtt106∆ on viability of rrm3∆ cells. Yeast strains of indicated genotypes were streaked onto YPD plates and grown at 30° for 3 days.
Figure 4
Figure 4
CTF4 deletion suppresses rrm3∆ lethality in different genetic contexts affecting the H3K56ac pathway. (A) CTF4 deletion rescues asf1∆ rrm3∆ lethality. Tetrads from diploids heterozygous for ctf4∆, rrm3∆, and asf1∆ were dissected and analyzed after 3 days at 30°. Circles indicate ctf4∆ asf1∆ rrm3∆ mutants. Dashed circle indicates asf1∆ rrm3∆ mutants. In A–E, diamonds indicate ctf4∆ rrm3∆ mutants. (B) CTF4 deletion rescues rtt109∆ rrm3∆ lethality. Tetrads from diploids heterozygous for ctf4∆, rrm3∆, and rtt109∆ were dissected and analyzed as in A. Circles indicate ctf4∆ rtt109∆ rrm3∆ mutants. Dashed circles indicate rtt109∆ rrm3∆ mutants. (C) CTF4 deletion rescues rtt101∆ rrm3∆ lethality. Tetrads from diploids heterozygous for ctf4∆, rrm3∆, and rtt101∆ were dissected and analyzed as in A. Circle indicates ctf4∆ rtt101∆ rrm3∆ mutant. Dashed circles indicate rtt101∆ rrm3∆ mutants. (D) CTF4 deletion rescues mms1∆ rrm3∆ lethality. Tetrads from diploids heterozygous for ctf4∆, rrm3∆, and mms1∆ were dissected and analyzed as in A. Circle indicates ctf4∆ mms1∆ rrm3∆ mutants. Dashed circles indicate mms1∆ rrm3∆ mutants. (E) CTF4 deletion partially rescues mms22∆ rrm3∆ lethality. Tetrads from diploids heterozygous for ctf4∆, rrm3∆, and mms22∆ were dissected and analyzed as in A. Circles indicate ctf4∆ mms22∆ rrm3∆ mutants. Dashed circles indicate mms22∆ rrm3∆ mutants.
Figure 5
Figure 5
In presence of MMS, CTF4 becomes harmful for cells affected in the H3K56 acetylation pathway. Fivefold serial dilutions of exponentially growing cells were spotted onto a YPD plate or 0.015% MMS plate and incubated at 30° for 3 days.
Figure 6
Figure 6
Uncoupling of MCM helicase and DNA polymerase-α favors cell viability during replicative stress in the absence of H3K56 acetylation. (A) The inability of Ctf4 to bind GINS and DNA polymerase-α restores the viability of the asf1∆ rrm3∆ mutant. Tetrad dissection from the ctf4-NT/CTF4 asf1∆/ASF1 rrm3∆/RRM3 diploid strain. Diamond, circle, and dashed circle indicate ctf4-NT rrm3∆, ctf4-NT asf1∆ rrm3∆, and asf1∆ rrm3∆ mutants, respectively. (B) The ability of Ctf4 to bind GINS and DNA polymerase-α is lethal in the asf1∆ rrm3∆ mutant. Diamond, circles, and dashed circle indicate ctf4-∆NT rrm3∆, ctf4-∆NT asf1∆ rrm3∆, and asf1∆ rrm3∆ mutants, respectively. (C) Sensitivity to CPT of the combination of the mutants ctf4∆, ctf4-NT, and ctf4-∆NT with asf1∆. Fivefold serials dilutions of exponentially growing cells were spotted onto YPD and 4µM CPT plates and incubated at 30° for 3 days. (D) Affecting the stability of the catalytic subunit of the DNA polymerase-α (Cdc17) restores the viability of asf1∆ rrm3∆ mutant. Tetrad dissection from cdc17-1/cdc17-1 asf1∆/ASF1 rrm3∆/RRM3 diploid strain. Circles indicate the asf1∆ rrm3∆ cdc17-1 mutants.
Figure 7
Figure 7
Ctf4 chromatin association is not affected in presence of replicative damages. (A) Cell cycle chromatin association of Ctf4. Ctf4-Myc cells were synchronized in G1 with α-factor and released into fresh medium at 25°. Samples were collected every 10 min, crude chromatin was prepared and analyzed by Western blot with 9E10 antibody for Ctf4-Myc (upper) and H3K56ac antibody for H3K56 acetylation (lower) using the same blot. Cell cycle progression was followed by FACS analysis (right). (B) Cell cycle chromatin association of Ctf4 in rrm3∆ cells. Ctf4-Myc rrm3∆ cells were treated and analyzed as in A. (C) Cell cycle chromatin association of Ctf4 in the presence of CPT. Ctf4-Myc cells were synchronized in G1 with α-factor and released in a new cell cycle at 25° in the presence of 40 µM of CPT. Samples were collected, prepared, and analyzed as in A. (D) The level of Ctf4 is reduced in hht1-K56R cells. ctf4-myc and ctf4-myc hht1∆-hhf1∆ hht2∆-hhf2∆ +phht1-K56R-HHF1 cells were synchronized in G1 with α-factor and released in a fresh medium at 30°. Samples were collected every 10 min and analyzed by Western blot with 9E10 antibody for Ctf4-Myc detection (upper). Anti-Rfa1 antibody was used as a loading control. Cell cycle progression was followed by FACS analysis (right).
Figure 8
Figure 8
Replicative stress induced by the absence of RRM3 increases Mms22 association with the replisome. (A) Pulldown protein extracts were loaded on Bis–Tris acrylamide gels in MOPS buffer and staked as a single band before trypsin digestion followed by mass spectrometry analysis. (Left) Mass spectroscopy data obtained after immunoprecipitation of Ctf4-GFP during S phase for Mms22, the catalytic subunit of the DNA polymerase-α (Cdc17), and MCM helicase. Spectral counts show the total number of identified peptide sequences for the indicated protein in each sample (RRM3 CTF4-GFP, rrm3∆ CTF4-GFP, and control CTF4). (Right) Relative quantitation of Mms22 protein compared to Ctf4 protein measured by the ratio of the sum of the areas of the three more intense precursor ions used for each protein identification. The averages of several independent experiments are shown. (B) The level of Mms22 is decreased in rrm3∆ cells. Protein extracts from mms22∆ + pG16adh-TAP-MMS22 and mms22∆ rrm3∆ + pG16adh-TAP-MMS22 strains were prepared from S-phase-synchronized cells and analyzed by Western blot with a protein-A antibody (right). Total proteins on the membrane were stained with Ponceau S as a loading control (left).
Figure 9
Figure 9
The replicative function of MRC1 is deleterious in asf1∆ cells experiencing replicative damages. (A) Effects of mrc1∆ on viability of the asf1∆ cells. Tenfold serial dilutions of exponentially growing cells were spotted onto YPD plates incubated at 30° or 38°, onto 5-µM CPT, and 0.01%-MMS plates incubated at 30° for 3 days. (B) Effects associated with the replicative and checkpoint functions of MRC1 on viability of the asf1∆ cells. Tenfold serial dilutions of exponentially growing cells were spotted onto YPD, 5-µM CPT, and 0.005%-MMS plates and incubated at 30° for 3 days.
Figure 10
Figure 10
H3K56 acetylation prevents genomic instability by affecting Ctf4 function. During S phase, in an unperturbed cell cycle (left), Asf1 functions cooperatively with Rtt109 to acetylate new histone H3 at lysine 56 (solid circles). The Rtt101–Mms1–Mms22 complex binds and ubiquitylates new H3K56ac histones (shaded circles) and promotes an efficient progression of the replication fork and nucleosome assembly by favoring H3-H4 transfer from Asf1 to other histone chaperones. At the end of S-phase, Hst3 and Hst4 deacetylases remove H3K56ac. How ubiquitylation is removed is still unknown. In response to DNA-damaging agents and in rrm3∆ cells (right), replication fork progression is affected, leading to checkpoint activation and subsequently to transcriptional repression of HST3 and HST4 and degradation of Hst3 and Hst4, allowing H3K56ac to persist. The unique chromatin environment created by H3K56ac accumulation behind the fork triggers a crucial interaction between Ctf4 and the Rtt101–Mms1–Mms22 complex through the N-terminal domain of Ctf4. This interaction modulates replisome structure by uncoupling MCM helicase and DNA polymerases and increases genome accessibility at replication defective forks, thus preserving genome integrity. It is currently unclear whether the interaction between Ctf4 and the Rtt101–Mms1–Mms22 complex is required to ubiquitylate Ctf4 itself and modulate its function or to recruit the Rtt101–Mms1–Mms22 complex to the site of DNA damage to ubiquitylate other replisome components. The function of the H3K56ac pathway is totally abolished in the absence of the N-terminal domain of Ctf4 required for its interaction with Mms22, positioning Ctf4 in the H3K56ac pathway.
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