Replisome Function During Replicative Stress Is Modulated by Histone H3 Lysine 56 Acetylation Through Ctf4

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 T HE eukaryotic replisome consists of polymerases and an essential DNA helicase that are linked by a number of factors assembled during the initiation of chromosome replication. Progression of the replication fork depends on the activity of the replisome progression complex (RPC). This complex is uniquely present during S phase (Gambus et al. 2006) and remains associated with the replication fork until completion of DNA replication. In Saccharomyces cerevisiae, the RPC is made up of Mcm10, Mrc1, Tof1, Csm3, Ctf4, Top1, FACT (Spt16 and Pob3), and the CMG complex comprising Cdc45, , and the go ichi ni san (GINS) complex. The CMG constitutes the core replicative helicase responsible for the movement and activities of the replication fork (Pacek et al. 2006;Bochman and Schwacha 2009).The link between helicase and polymerases is a crucial determinant for the regulation of the replisome. The leadingstrand DNA polymerase-ɛ was recently shown to be integrated into the replisome via an interaction with the GINS complex (Sengupta et al. 2013). Furthermore, the DNA polymerasea-primase complex, which initiates DNA synthesis at replication origins and continues to prime Okazaki fragments at the fork, remains associated with the RPC via the Ctf4 trimer, which simultaneously interacts with the GINS complex (Gambus et al. 2009;Tanaka et al. 2009;Gosnell and Christensen 2011;Simon et al. 2014).Cells have evolved different mechanisms to maintain genome integrity under the conditions threatening replication progression (Jossen and Bermejo 2013;Leman and Noguchi 2013). The S-phase checkpoint mediated by MRC1 was initially characterized as a pathway activated by fork stalling and able to both stabilize the replisome and dela...

Fission Yeast Sirtuin Hst4 Functions in Preserving Genomic Integrity by Regulating Replisome Component Mcl1

“…In S . cerevisiae , it has been shown that Mcl1 homologue Ctf4 is a major target of H3K56ac pathway 44 . Our data showed that Mcl1 expression could suppress hst4 Δ mutant phenotypes.…”

The Replisome-Coupled E3 Ubiquitin Ligase Rtt101Mms22 Counteracts Mrc1 Function to Tolerate Genotoxic Stress

“…We next examined how the Rtt101 Mms22 E3 ligase is recruited to active replisomes. Ctf4 was an intriguing candidate as it was previously found to interact with Mms22 [ 3 , 18 , 36 ]. Indeed, genetic analysis revealed that the growth defect of ctf4Δ cells on genotoxic drugs was epistatic with RTT101 and even slightly suppressed the sensitivity of mms22Δ cells ( Fig 3A ), consistent with previous findings [ 36 ].…”

“…Conversely, the replication fork progression defect is enhanced in mrc1Δ compared to tof1Δ and csm3Δ mutants [ 1 ], implying that Mrc1 also promotes replication functions independent of Tof1 and Csm3. Ctf4, the yeast homologue of human AND1, bridges the interaction of the primase, DNA polymerase-α, to the CMG helicase [ 3 , 5 , 18 ]. Although the coupling of the CMG to leading and lagging strand DNA polymerases preserves genome integrity during unperturbed DNA replication, this mechanism is partially disrupted when forks encounter replication stress.…”

“…However, the underlying mechanisms and function of the Rtt101-Mms1-Mms22 complex (termed Rtt101 Mms22 ) remain largely elusive. Recent results suggest that the sensitivity of mms1Δ , mms22Δ and rtt101Δ cells to the DNA-damaging drugs CPT and MMS could be rescued by further deleting MRC1 [ 18 , 29 ], possibly by de-regulating late firing origins [ 29 ].…”

Histone H4 acetylation required for chromatin decompaction during DNA replication

“…Analysis of replication fork dynamics in living cells also suggests that the sites of DNA synthesis are sites of decondensed chromatin 4 . Posttranslational modification of histones, such as acetylation of histones H3 and H4, has been reported to regulate DNA replication 5 6 7 probably by affecting chromatin structures 8 . However, the molecular mechanisms underlying the DNA replication-associated alteration of chromatin structures remain unknown.…”