Histone H3 K56 hyperacetylation perturbs replisomes and causes DNA damage

Abstract

Deacetylation of histone H3 K56, regulated by the sirtuins Hst3p and Hst4p, is critical for maintenance of genomic stability. However, the physiological consequences of a lack of H3 K56 deacetylation are poorly understood. Here we show that cells lacking Hst3p and Hst4p, in which H3 K56 is constitutively hyperacetylated, exhibit hallmarks of spontaneous DNA damage, such as activation of the checkpoint kinase Rad53p and upregulation of DNA-damage inducible genes. Consistently, hst3 hst4 cells display synthetic lethality interactions with mutations that cripple genes involved in DNA replication and DNA double-strand break (DSB) repair. In most cases, synthetic lethality depends upon hyperacetylation of H3 K56 because it can be suppressed by mutation of K56 to arginine, which mimics the nonacetylated state. We also show that hst3 hst4 phenotypes can be suppressed by overexpression of the PCNA clamp loader large subunit, Rfc1p, and by inactivation of the alternative clamp loaders CTF18, RAD24, and ELG1. Loss of CTF4, encoding a replisome component involved in sister chromatid cohesion, also suppresses hst3 hst4 phenotypes. Genetic analysis suggests that CTF4 is a part of the K56 acetylation pathway that converges on and modulates replisome function. This pathway represents an important mechanism for maintenance of genomic stability and depends upon proper regulation of H3 K56 acetylation by Hst3p and Hst4p. Our data also suggest the existence of a precarious balance between Rfc1p and the other RFC complexes and that the nonreplicative forms of RFC are strongly deleterious to cells that have genomewide and constitutive H3 K56 hyperacetylation.

Figure 1.—

Overexpression of RFC1 suppresses the growth defect, Ts phenotype, and sensitivity to genotoxic agents of hst3 hst4 cells. (A) Serial dilutions (1:5) of strains ICY188 (WT + YEP351), ICY189 (WT + YEP351/RFC1), ICY190 (hst3 hst4 + YEP351), and ICY191 (hst3 hst4 + YEP351/RFC1) were spotted on SC −Leu −Ura and SC −Leu +5-FOA and grown at the indicated temperatures for 2–3 days. (B) After shuffling out a URA3-marked plasmid on 5-FOA and an additional round of 5-FOA selection, the strains ICY188, ICY190, and ICY191 were spotted in serial dilutions (1:5) on SC −Leu and grown at 30° as indicated.

Figure 2.—

Suppression of the hst3 hst4 growth defect and Ts phenotype by inactivation of alternative RFC complexes. (A) Serial dilutions (1:5) of strains ICY356 (WT), ICY703 (hst3 hst4), ICY1528 (hst3 hst4 rad24), ICY1534 (hst3 hst4 elg1), ICY1537 (hst3 hst4 ctf18), ICY1544 (hst3 hst4 mec3), ICY1550 (hst3 hst4 rad17), ICY1556 (hst3 hst4 ddc1), ICY1797 (hst3 hst4 ctf8), ICY1795 (hst3 hst4 dcc1) were spotted on SC −Ura or SC +5-FOA and grown for 3 days at the indicated temperatures. (B) Serial dilutions (1:5) of strains ICY356 (WT), ICY252 (hst3 hst4), ICY342 (hst3 hst4 rad24), ICY449 (hst3 hst4 rad9), and ICY773 (hst3 hst4 rad9 rad24) were spotted on SC −Ura (25°), SC +5-FOA (25°), and SC +5-FOA (37°) and grown for 3 days at the indicated temperatures.

Figure 3.—

Genetic interaction with ctf4 and ctf18. (A) Suppression of the hst3 hst4 Ts phenotype and HU sensitivity by deletion of CTF4. Serial dilutions (1:5) of strains ICY1488 (WT), ICY1676 (ctf4), ICY1514 (hst3 hst4), and ICY1684 (hst3 hst4 ctf4) were spotted on YPD and YPD + 100 mm HU and grown for 3 days at 25° and 37° (YPD) and 5 days at 25° (YPD + HU). (B) Analysis of genetic interaction between ctf4, ctf18, and hht1(K56R). Serial dilutions (1:5) of strains ICY1492 (hht2-hhf2), ICY1688 (hht2-hhf2 ctf4), ICY1605 (hht2-hhf2 ctf18), ICY1497 [hht2-hhf2 hht1(K56R)], ICY1692 [hht2-hhf2 hht1(K56R) ctf4], and ICY1607 [hht2-hhf2 hht1(K56R) ctf18] were spotted on YPD, YPD + 50 mm HU, and YPD + 1 μg/ml CPT and grown for 3 days (YPD) and 5 days (YPD + HU; YPD + CPT) at 25°.

Figure 4.—

Analysis of K56 acetylation. (A) Total protein extracts were prepared from strains ICY188 (WT + YEP351), ICY190 (hst3 hst4 + YEP351), ICY191 (hst3 hst4 + YEP351/RFC1), and ICY192 (hst3 hst4 + YEP351/RFC1) after two rounds of 5-FOA selection to lose the URA3 (WT) and URA3-HST3 (hst3 hst4 strains) plasmids, and the acetylation of histone H3 K56 was analyzed by immunoblotting with a K56Ac-specific antibody. The membrane was stripped and reprobed with an antibody specific for the C terminus of H3. (B) Total protein extracts from strains ICY1488 (WT), ICY1514 (hst3 hst4), ICY1646 (hst3 hst4 rad24), ICY1664 (hst3 hst4 elg1), ICY1613 (hst3 hst4 ctf18), ICY1684 (hst3 hst4 ctf4), and ICY1216 (asf1) were separated by SDS–PAGE and immunoblotted with a K56Ac specific antibody. The membrane was stripped and reprobed with an antibody specific for the C terminus of H3.

Figure 5.—

Like wild-type cells, hst3 hst4 cells slow down DNA replication and spindle elongation when exposed to MMS and HU, respectively. (A) ICY48 (bar1) and ICY49 (bar1 hst3 hst4) cells were arrested with α-factor and released into medium with and without MMS. Aliquots of the cells were taken at indicated time points and analyzed by FACS. (B–C) ICY48 (bar1) and ICY49 (bar1 hst3 hst4) cells were arrested with α-factor and released into medium with and without 100 mm HU. Aliquots of the cells were taken at indicated time points and analyzed by FACS (B) and immunofluorescence for tubulin staining (C).

Figure 6.—

Induction of RNR3 and HUG1 and hyperphosphorylation of Rad53p in hst3 hst4 cells. (A) Total RNA was isolated from strains YCB617 (WT), YCB470 (hst3), YCB575 (hst4), ICY252 (hst3 hst4), ICY342 (hst3 hst4 rad24), ICY449 (hst3 hst4 rad9), and ICY773 (hst3 hst4 rad9 rad24) and hybridized to RNR3, HUG1, and actin-specific probes. Prior to the experiment, strains ICY252, 342, 449, and 773 were grown on 5-FOA to shuffle out a URA3 plasmid carrying the HST3 gene. (B) Rad53p is hyperphosphorylated in a Rad24p- and Rad9p-dependent manner in hst3 hst4 cells, but not in the single mutants. Total protein extracts from the strains YCB617 (WT), YCB470 (hst3), YCB575 (hst4), ICY252 (hst3 hst4), ICY342 (hst3 hst4 rad24), ICY449 (hst3 hst4 rad9), and ICY773 (hst3 hst4 rad9 rad24) were separated by SDS–PAGE and immunoblotted with an antibody specific for Rad53p. Prior to the experiment, strains ICY252, 342, 449, and 773 were grown on 5-FOA to shuffle out a URA3 plasmid carrying the HST3 gene. (C) H3 K56R mutation reduces Rad53p phosphorylation in hst3 hst4 strains. Total protein extracts were prepared from strains ICY1488 (WT), ICY1514 (hst3 hst4), ICY1518 (hst3 hst4 hht1K56R), ICY1492 (hht2-hhf2), ICY1497 (hht2-hhf2 hht1K56R), ICY1501 (hht2-hhf2 hst3 hst4), and ICY1506 (hht2-hhf2 hht1K56R hst3 hst4) and analyzed for Rad53p phosphorylation using a Rad53p-specific antibody.

Figure 7.—

hst3 hst4 cells require a Mec1p-dependent function for viability. (A) Synthetic lethality between hst3 hst4 and pol2-11. Serial dilutions (1:5) of strains ICY356 (WT), ICY674 (pol2-11), ICY676 (pol2-11 hst3), ICY680 (pol2-11 hst4), ICY610 (hst3 hst4), and ICY682 (pol2-11 hst3 hst4) were spotted on SC −Ura and SC +FOA and grown for 3–4 days at 25°. (B) Synthetic lethality between hst3 hst4 and mec1 and dun1. Serial dilutions (1:5) of strains ICY356 (WT), ICY703 (hst3 hst4), ICY430 (hst3 hst4 sml1), ICY431 (hst3 hst4 sml1 mec1), ICY351 (hst3 hst4 sml1 rad53), and ICY230 (hst3 hst4 dun1) were spotted on SC −Ura and SC +FOA and grown for 3–4 days at 30°.

Figure 8.—

Synthetic lethality analysis with hst3 hst4 and suppression with H3 K56R. (A) Serial dilutions (1:5) of strains ICY356a (WT + vector), ICY703a (hst3 hst4 + vector), ICY703b (hst3 hst4 + HHT1), and ICY703c (hst3 hst4 + hht1K56R) were spotted on SC −Leu −Ura and SC −Leu +5-FOA and grown at the indicated temperatures for 2 and 3 days, respectively. (B) Serial dilutions (1:5) of strains ICY356a (WT + vector), ICY703a (hst3 hst4 + vector), ICY431a, -b, and -c, ICY1566a, -b, and -c, ICY1568a, -b, and -c, ICY1570a, -b, and -c, ICY1572a, -b, and -c, ICY682a, -b, and -c, ICY1574a, -b, and -c, and ICY1576a, -b, and -c were spotted on SC −Leu −Ura and SC −Leu +5-FOA and grown at the indicated temperatures for 2 and 3 days, respectively.