Cdc48 and a ubiquitin ligase drive disassembly of the CMG helicase at the end of DNA replication

Marić, Marija; Maculins, Timurs; Piccoli, Giacomo De; Labib, Karim

doi:10.1126/science.1253596

Cited by 204 publications

(336 citation statements)

References 56 publications

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“…The arrested fork, with the help of DNA polymerase, helicase, and topoisomerase (5), merges with the fork approaching Ter from the opposite direction, and the resulting catenated daughter molecules are resolved by topoisomerase IV in bacteria (6) and topoisomerase II in eukaryotes (7,8). In budding yeast, the unloading of the replisomal components from the template after replication termination is facilitated by Cdc48 and a ubiquitin ligase (9).…”

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confidence: 99%

Phosphorylation of CMG helicase and Tof1 is required for programmed fork arrest

Bastia

Srivastava

Zaman

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Several important physiological transactions, including control of replicative life span (RLS), prevention of collision between replication and transcription, and cellular differentiation, require programmed replication fork arrest (PFA). However, a general mechanism of PFA has remained elusive. We previously showed that the Tof1–Csm3 fork protection complex is essential for PFA by antagonizing the Rrm3 helicase that displaces nonhistone protein barriers that impede fork progression. Here we show that mutations of Dbf4-dependent kinase (DDK) of Saccharomyces cerevisiae, but not other DNA replication factors, greatly reduced PFA at replication fork barriers in the spacer regions of the ribosomal DNA array. A key target of DDK is the mini chromosome maintenance (Mcm) 2–7 complex, which is known to require phosphorylation by DDK to form an active CMG [Cdc45 (cell division cycle gene 45), Mcm2–7, GINS (Go, Ichi, Ni, and San)] helicase. In vivo experiments showed that mutational inactivation of DDK caused release of Tof1 from the chromatin fractions. In vitro binding experiments confirmed that CMG and/or Mcm2–7 had to be phosphorylated for binding to phospho-Tof1–Csm3 but not to its dephosphorylated form. Suppressor mutations that bypass the requirement for Mcm2–7 phosphorylation by DDK restored PFA in the absence of the kinase. Retention of Tof1 in the chromatin fraction and PFA in vivo was promoted by the suppressor mcm5-bob1, which bypassed DDK requirement, indicating that under this condition a kinase other than DDK catalyzed the phosphorylation of Tof1. We propose that phosphorylation regulates the recruitment and retention of Tof1–Csm3 by the replisome and that this complex antagonizes the Rrm3 helicase, thereby promoting PFA, by preserving the integrity of the Fob1–Ter complex.

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mentioning

confidence: 99%

Phosphorylation of CMG helicase and Tof1 is required for programmed fork arrest

Bastia

Srivastava

Zaman

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…During replication termination, CMG unloading was shown to involve polyubiquitylation of MCM7 by using K48-linked chains (33,34). To compare p97-mediated helicase unloading during ICL repair and replication termination, pICL or an undamaged control plasmid (pControl) was allowed to replicate in extract.…”

Section: Resultsmentioning

confidence: 99%

“…3D, 4, and 5B) (33,34). Although K48 linkages play a prominent role in protein turnover, proteasomal degradation was not required for helicase unloading (Fig.…”

Section: Discussionmentioning

confidence: 99%

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p97 Promotes a Conserved Mechanism of Helicase Unloading during DNA Cross-Link Repair

Fullbright

Rycenga

Gruber

et al. 2016

Molecular and Cellular Biology

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Interstrand cross-links (ICLs) are extremely toxic DNA lesions that create an impassable roadblock to DNA replication. When a replication fork collides with an ICL, it triggers a damage response that promotes multiple DNA processing events required to excise the cross-link from chromatin and resolve the stalled replication fork. One of the first steps in this process involves displacement of the CMG replicative helicase (comprised of Cdc45, MCM2-7, and GINS), which obstructs the underlying crosslink. Here we report that the p97/Cdc48/VCP segregase plays a critical role in ICL repair by unloading the CMG complex from chromatin. Eviction of the stalled helicase involves K48-linked polyubiquitylation of MCM7, p97-mediated extraction of CMG, and a largely degradation-independent mechanism of MCM7 deubiquitylation. Our results show that ICL repair and replication termination both utilize a similar mechanism to displace the CMG complex from chromatin. However, unlike termination, repair-mediated helicase unloading involves the tumor suppressor protein BRCA1, which acts upstream of MCM7 ubiquitylation and p97 recruitment. Together, these findings indicate that p97 plays a conserved role in dismantling the CMG helicase complex during different cellular events, but that distinct regulatory signals ultimately control when and where unloading takes place. Interstrand cross-links (ICLs) are extremely toxic DNA lesions that covalently couple both strands of the DNA duplex. Replicating cells are particularly sensitive to ICLs, which disrupt the strand separation required for DNA replication and transcription (1). Repair of ICLs is initiated primarily during S phase when a replication fork collides with the cross-link (2-4). Fork stalling triggers a damage response that involves multiple DNA processing events that promote excision of the ICL from chromatin and resolution of the stalled replication fork (5). Defects in ICL repair are associated with delays in cell cycle progression, increased chromosomal breakage, and severe sensitivity to DNA cross-linking agents. The cellular symptoms of deficient or aberrant repair are thought to underlie the molecular basis of several cancer predisposition syndromes (6), including Fanconi anemia (FA) and hereditary breast and ovarian cancer.Fanconi anemia is a rare chromosomal instability disorder caused by mutations in 1 of at least 16 different genes (7). Four of the FA genes are classified as breast/ovarian cancer susceptibility (BRCA) genes (FANCD1/BRCA2, FANCJ/BRIP1, FANCN/ PALB2, FANCO/RAD51C) (8), with recent evidence also supporting classification of BRCA1 as a new FA subtype (FANCS) (9). Together, the FA/BRCA proteins and associated factors form a multifunctional network that plays a critical role in managing various forms of DNA damage and replication stress (10). Although both FA and BRCA proteins are required for ICL repair, each pathway also supports nonoverlapping functions (11, 12) that play different roles in how cells respond to treatment with DNA cross-linking agents.Xenop...

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“…A recent study has shown that Schizosaccharomyces pombe cells elicit a program to degrade replisome upon DNA replication stress through the action of the ubiquitin-proteasome system SCF Pof3 , a homolog of the budding yeast SCF Dia2 ubiquitin-proteasome system (Roseaulin et al 2013a,b). Because Ctf4 physically interacts with Dia2, which drives CMG helicase and replisome disassembly at the end of DNA replication (Maric et al 2014;Moreno et al 2014) and is ubiquitylated by SCF Dia2 (Ho et al 2002;Collins et al 2007;Mimura et al 2009;Morohashi et al 2009), one possibility could be that Ctf4 is targeted by Dia2 in response to replicative stress in a way dependent on H3K56ac. However, because the ctf4-DNT mutation that preserves the Ctf4 interaction with Dia2 (Mimura et al 2009;Morohashi et al 2009) does not restore the viability of asf1D rrm3D cells and is lethal in the rrm3D mutant (in contrast to the ctf4-NT mutation that loses the Ctf4-Dia2 interaction), we exclude the possibility that Ctf4 action could be regulated during replicative stress by H3K56ac through an action mediated by the SCF Dia2 .…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2015

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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...

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Cdc48 and a ubiquitin ligase drive disassembly of the CMG helicase at the end of DNA replication

Cited by 204 publications

References 56 publications

Phosphorylation of CMG helicase and Tof1 is required for programmed fork arrest

Phosphorylation of CMG helicase and Tof1 is required for programmed fork arrest

p97 Promotes a Conserved Mechanism of Helicase Unloading during DNA Cross-Link Repair

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

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