Licensing for DNA replication requires a strict sequential assembly of Cdc6 and Cdt1 onto chromatin in Xenopus egg extracts

Tsuyama, Takashi; Tada, Shusuke; Watanabe, Satoshi; Seki, Masayuki; Enomoto, Takemi

doi:10.1093/nar/gki226

Cited by 37 publications

(47 citation statements)

References 47 publications

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“…This idea is in agreement with Palacios DeBeer et al (2003), who demonstrate that origin and HM silencer ACS differ in their affinity for the ORC and that this difference reflects the preferential function of the corresponding ACS in silencing or origin firing. We propose that an additional functional dissimilarity of these two types of ACS is their dependence on the cdc6-1 allele (Figure 3) (Weinreich et al 2001;Tsuyama et al 2005). It is possible that the surrounding sequence of core X-and Y9-elements influences the ACS silencing specificity.…”

Section: Discussionmentioning

confidence: 94%

“…Cdc6p is essential for the loading of the MCM complex on ORC-bound ACS at origins of replication (Blow and Dutta 2005). The cdc6-1 allele impairs DNA replication (Weinreich et al 2001;Tsuyama et al 2005). However, it selectively caused derepression only in the ACS-less GF2 and GF3 constructs, while having negligible effects on the ACS-containing constructs ( Figure 3G).…”

Section: Discussionmentioning

confidence: 99%

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Differential Requirement of DNA Replication Factors for Subtelomeric ARS Consensus Sequence Protosilencers in Saccharomyces cerevisiae

et al. 2006

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The establishment of silent chromatin requires passage through S-phase, but not DNA replication per se. Nevertheless, many proteins that affect silencing are bona fide DNA replication factors. It is not clear if mutations in these replication factors affect silencing directly or indirectly via deregulation of S-phase or DNA replication. Consequently, the relationship between DNA replication and silencing remains an issue of debate. Here we analyze the effect of mutations in DNA replication factors (mcm5-461, mcm5-1, orc2-1, orc5-1, cdc45-1, cdc6-1, and cdc7-1) on the silencing of a group of reporter constructs, which contain different combinations of ''natural'' subtelomeric elements. We show that the mcm5-461, mcm5-1, and orc2-1 mutations affect silencing through subtelomeric ARS consensus sequences (ACS), while cdc6-1 affects silencing independently of ACS. orc5-1, cdc45-1, and cdc7-1 affect silencing through ACS, but also show ACSindependent effects. We also demonstrate that isolated nontelomeric ACS do not recapitulate the same effects when inserted in the telomere. We propose a model that defines the modes of action of MCM5 and CDC6 in silencing.

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Section: Discussionmentioning

confidence: 94%

Section: Discussionmentioning

confidence: 99%

Differential Requirement of DNA Replication Factors for Subtelomeric ARS Consensus Sequence Protosilencers in Saccharomyces cerevisiae

et al. 2006

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“…To address GST-Cdt1-induced loading of replication-related proteins onto overreplicating chromatin, sperm nuclei (22,000 nuclei) were incubated with egg extracts (18 l) for 90 min, the reaction mixture was supplemented with GST-Cdt1, caffeine, and/or Gem79-130, and the reaction volume was adjusted to 22 l. The samples were further incubated for 30 or 90 min. The resulting chromatin fractions were isolated and immunoblotted as described previously (Tsuyama et al, 2005).To examine the phosphorylation of Chk1 by immunoblotting, nuclei in egg extracts were isolated as described previously (Kumagai et al, 1998), with a slight modification. After a 90-min incubation of Xenopus egg extracts with sperm nuclei (34,000 nuclei in 29 l), the reaction mixture was further incubated for 90 min with 5 l of buffer or GST-Cdt1 in the presence or absence of caffeine or Gem79-130.…”

mentioning

confidence: 99%

Repression of Nascent Strand Elongation by Deregulated Cdt1 during DNA Replication inXenopusEgg Extracts

Tsuyama

Watanabe

Aoki

et al. 2009

MBoC

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Excess Cdt1 reportedly induces rereplication of chromatin in cultured cells and Xenopus egg extracts, suggesting that the regulation of Cdt1 activity by cell cycle-dependent proteolysis and expression of the Cdt1 inhibitor geminin is crucial for the inhibition of chromosomal overreplication between S phase and metaphase. We analyzed the consequences of excess Cdt1 for DNA replication and found that increased Cdt1 activity inhibited the elongation of nascent strands in Xenopus egg extracts. In Cdt1-supplemented extracts, overreplication was remarkably induced by the further addition of the Cdt1-binding domain of geminin (Gem79-130), which lacks licensing inhibitor activity. Further analyses indicated that fully active geminin, as well as Gem79-130, restored nascent strand elongation in Cdt1-supplemented extracts even after the Cdt1-induced stalling of replication fork elongation had been established. Our results demonstrate an unforeseen, negative role for Cdt1 in elongation and suggest that its function in the control of replication should be redefined. We propose a novel surveillance mechanism in which Cdt1 blocks nascent chain elongation after detecting illegitimate activation of the licensing system. INTRODUCTIONTo maintain genome integrity, chromosomes are precisely duplicated only once per cell division cycle. In eukaryotic cells, the replication licensing system ensures accurate DNA replication. The prereplication complex associates with origins of replication before S phase through the stepwise assembly of the origin recognition complex, Cdc6, Cdt1, and Mcm2-7, after which licensing takes place (Diffley, 2004;Blow and Dutta, 2006). Mcm2-7 is thought to act as the replicative helicase, and the loading of Mcm2-7 onto chromatin is considered to be the key initiating event of the licensing reaction (Pacek and Walter, 2004). Licensed origins are presumably activated in S phase by Cdc7-and cyclindependent kinase (CDK)-dependent processes, leading to the formation of replication forks and the recruitment of DNA polymerases.Repression of licensing after the onset of S phase is crucial for preventing rereplication (Fujita, 2006;Arias and Walter, 2007). In fission yeast, overexpression of Cdc18, an orthologue of Cdc6 in budding yeast and higher eukaryotes, induces rereplication (Nishitani et al., 2000;Yanow et al., 2001), suggesting that Cdc18/Cdc6 is a major target of mechanisms that repress licensing. In contrast, Cdt1 seems to be the crucial target in higher eukaryotes, because unregulated Cdt1 activity alone induces rereplication (Vaziri et al., 2003;Nishitani et al., 2004;Li and Blow, 2005;Arias and Walter, 2005;Maiorano et al., 2005). Geminin represses licensing by binding and inhibiting Cdt1 (Wohlschlegel et al., 2000;Tada et al., 2001), and it is inactivated or degraded on mitotic exit (McGarry and Kirschner, 1998;Li and Blow, 2004). The nuclear import of geminin during S phase reactivates it to restrict licensing (Hodgson et al., 2002;Yoshida et al., 2005). Crystallographic analysis revealed that geminin f...

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“…In contrast, elimination of Cdc6 interferes with Cdt1 origin binding in vitro (Randell et al 2006;). Studies in Xenopus extracts have shown that Cdt1 associates with chromatin in the absence of Cdc6 (Gillespie et al 2001;Tsuyama et al 2005); however, only Cdt1 associated in the presence of Cdc6 is able to contribute to Mcm2 -7 loading (Tsuyama et al 2005). Consistent with a robust interaction between Cdc6 and ORC, a complex between the proteins has been structurally characterized (Speck et al 2005;Sun et al 2012) and origin-bound ORC stimulates Cdc6 ATP hydrolysis (Randell et al 2006).…”

Section: Origin Recognitionmentioning

confidence: 98%

“…Biochemical studies support a model in which ORC first interacts with Cdc6 and this complex then recruits Cdt1 and Mcm2 -7. Loss of Cdt1 does not prevent Cdc6 chromatin association in vivo or origin recruitment in vitro (Maiorano et al 2000;Nishitani et al 2000;Tsuyama et al 2005;Randell et al 2006;). In contrast, elimination of Cdc6 interferes with Cdt1 origin binding in vitro (Randell et al 2006;).…”

Section: Origin Recognitionmentioning

confidence: 99%

Helicase Loading at Chromosomal Origins of Replication

Bell

Kaguni

2013

Cold Spring Harbor Perspectives in Biology

120

109

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Loading of the replicative DNA helicase at origins of replication is of central importance in DNA replication. As the first of the replication fork proteins assemble at chromosomal origins of replication, the loaded helicase is required for the recruitment of the rest of the replication machinery. In this work, we review the current knowledge of helicase loading at Escherichia coli and eukaryotic origins of replication. In each case, this process requires both an origin recognition protein as well as one or more additional proteins. Comparison of these events shows intriguing similarities that suggest a similar underlying mechanism, as well as critical differences that likely reflect the distinct processes that regulate helicase loading in bacterial and eukaryotic cells.

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Licensing for DNA replication requires a strict sequential assembly of Cdc6 and Cdt1 onto chromatin in Xenopus egg extracts

Cited by 37 publications

References 47 publications

Differential Requirement of DNA Replication Factors for Subtelomeric ARS Consensus Sequence Protosilencers in Saccharomyces cerevisiae

Differential Requirement of DNA Replication Factors for Subtelomeric ARS Consensus Sequence Protosilencers in Saccharomyces cerevisiae

Repression of Nascent Strand Elongation by Deregulated Cdt1 during DNA Replication inXenopusEgg Extracts

Helicase Loading at Chromosomal Origins of Replication

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