Characterization of the Xenopus replication licensing system

Chong, J.; Thömmes, Pia; Rowles, Alison; Mahbubani, Hiro; Blow, J. Julian

doi:10.1016/s0076-6879(97)83043-1

Cited by 75 publications

(151 citation statements)

References 25 publications

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“…Xenopus egg extracts (low-speed supernatant) were prepared as described previously (15). Immunodepletion was performed by mixing egg extracts three times with the antibody-crosslinked matrix at 4°C.…”

Section: Methodsmentioning

confidence: 99%

“…Immunodepletion was performed by mixing egg extracts three times with the antibody-crosslinked matrix at 4°C. DNA replication with membrane-removed sperm nuclei (2,000 sperm heads per l of extract) was carried out at 23°C in the presence of [␣-32 P]dATP as described elsewhere (15). The reaction products were purified by RNase A digestion, proteinase K digestion, and phenol͞chloroform extraction followed by ethanol precipitation and then separated by 0.8% agarose gel electrophoresis under neutral (Tris͞borate͞EDTA buffer) or alkaline (30 mM Abbreviations: Pol␣, -␦, and -, DNA polymerases ␣, ␦, and ; SV40, simian virus 40; RPA, replication protein A.…”

Section: Methodsmentioning

confidence: 99%

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DNA polymerase ɛ is required for coordinated and efficient chromosomal DNA replication in Xenopus egg extracts

Waga

Masuda

Takisawa

et al. 2001

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

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DNA polymerase (Pol) is thought to be involved in DNA replication, repair, and cell-cycle checkpoint control in eukaryotic cells. Although the requirement of other replicative DNA polymerases, DNA polymerases ␣ and ␦ (Pol␣ and ␦), for chromosomal DNA replication has been well documented by genetic and biochemical studies, the precise role, if any, of Pol in chromosomal DNA replication is still obscure. Here we show, with the use of a cell-free replication system with Xenopus egg extracts, that Xenopus Pol is indeed required for chromosomal DNA replication. In Poldepleted extracts, the elongation step of chromosomal DNA replication is markedly impaired, resulting in significant reduction of the overall DNA synthesis as well as accumulation of small replication intermediates. Moreover, despite the decreased DNA synthesis, excess amounts of Pol␣ are loaded onto the chromatin template in Pol-depleted extracts, indicative of the failure of proper assembly of DNA synthesis machinery at the fork. These findings strongly suggest that Pol, along with Pol␣ and Pol␦, is necessary for coordinated chromosomal DNA replication in eukaryotic cells.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

DNA polymerase ɛ is required for coordinated and efficient chromosomal DNA replication in Xenopus egg extracts

Waga

Masuda

Takisawa

et al. 2001

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

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“…Metaphase-arrested Xenopus egg extracts and demembranated Xenopus sperm nuclei were prepared as described previously (Chong et al, 1997). Egg extracts were used after release into interphase after incubation with 0.3 mM CaCl 2 for 15 min at 23°C.…”

Section: Preparation Of Xenopus Egg Extractsmentioning

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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“…DNA replication assays were carried out according to Chong et al (1997). Briefly, interphase LSS was depleted using control IgG, anti-XISWI, anti-XACF1, or anti-XWSTF antibody and supplemented with [␣-32 P]dATP (specific radioactivity of 3000 Ci/mmol) at a final concentration of 0.05 mCi/ml.…”

Section: Dna Replication Assaymentioning

confidence: 99%

ISWI Remodeling Complexes inXenopusEgg Extracts: Identification as Major Chromosomal Components that Are Regulated by INCENP-aurora B

MacCallum

Losada

Kobayashi

et al. 2002

MBoC

131

101

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We previously characterized major components of mitotic chromosomes assembled in Xenopus laevis egg extracts and collectively referred to them as Xenopus chromosome-associated polypeptides (XCAPs). They included five subunits of the condensin complex essential for chromosome condensation. In an effort to identify novel proteins involved in this process, we have isolated XCAP-F and found it to be the Xenopus ortholog of ISWI, a chromatin remodeling ATPase. ISWI exists in two major complexes in Xenopus egg extracts. The first complex contains ACF1 and two low-molecular-weight subunits, most likely corresponding to Xenopus CHRAC. The second complex is a novel one that contains the Xenopus ortholog of the human Williams syndrome transcription factor (WSTF). In the absence of the ISWI complexes, the deposition of histones onto DNA is apparently normal, but the spacing of nucleosomes is greatly disturbed. Despite the poor spacing of nucleosomes, ISWI depletion has little effect on DNA replication, chromosome condensation or sister chromatid cohesion in the cell-free extracts. The association of ISWI with chromatin is cell cycle regulated and is under the control of the INCENP-aurora B kinase complex that phosphorylates histone H3 during mitosis. Apparently contradictory to the generally accepted model, we find that neither chromosome condensation nor chromosomal targeting of condensin is compromised when H3 phosphorylation is drastically reduced by depletion of INCENP-aurora B.

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Characterization of the Xenopus replication licensing system

Cited by 75 publications

References 25 publications

DNA polymerase ɛ is required for coordinated and efficient chromosomal DNA replication in Xenopus egg extracts

DNA polymerase ɛ is required for coordinated and efficient chromosomal DNA replication in Xenopus egg extracts

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

ISWI Remodeling Complexes inXenopusEgg Extracts: Identification as Major Chromosomal Components that Are Regulated by INCENP-aurora B

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