Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1999 Jun;112 ( Pt 12)(Pt 12):2011-8.
doi: 10.1242/jcs.112.12.2011.

Changes in association of the Xenopus origin recognition complex with chromatin on licensing of replication origins

A Rowles  1 , S Tada, J J Blow
Affiliations

Changes in association of the Xenopus origin recognition complex with chromatin on licensing of replication origins

A Rowles et al. J Cell Sci. 1999 Jun.

Abstract

During late mitosis and early G1, a series of proteins are assembled onto replication origins that results in them becoming 'licensed' for replication in the subsequent S phase. In Xenopus this first involves the assembly onto chromatin of the Xenopus origin recognition complex XORC, and then XCdc6, and finally the RLF-M component of the replication licensing system. In this paper we examine changes in the way that XORC associates with chromatin in the Xenopus cell-free system as origins become licensed. Restricting the quantity of XORC on chromatin reduced the extent of replication as expected if a single molecule of XORC is sufficient to specify a single replication origin. During metaphase, XOrc1 associated only weakly with chromatin. In early interphase, XOrc1 formed a strong complex with chromatin, as evidenced by its resistance to elution by 200 mM salt, and this state persisted when XCdc6 was assembled onto the chromatin. As a consequence of origins becoming licensed the association of XOrc1 and XCdc6 with chromatin was destabilised, and XOrc1 became susceptible to removal from chromatin by exposure to either high salt or high Cdk levels. At this stage the essential function for XORC and XCdc6 in DNA replication had already been fulfilled. Since high Cdk levels are required for the initiation of DNA replication, this 'licensing-dependent origin inactivation' may contribute to mechanisms that prevent re-licensing of replication origins once S phase has started.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Effect on replication of limiting XORC. Xenopus sperm nuclei were incubated for 15 minutes in different mixtures of extract depleted with antibodies against XOrc1 (“XORC1−”) or non-immune antibodies (“NI−”). Chromatin was isolated and then either A, subjected to SDS - PAGE alongside known quantities of recombinant XOrc1 and immunoblotted with antibodies to XOrc1; or B, incubated for 140 minutes with [α-32P]dATP in XOrc1-depleted extract and the total amount of DNA synthesised determined. DNA synthesis is expressed as a percentage of that obtained in extract immunodepleted with non-immune antibodies. C. The quantities of XOrc1 present on the chromatin samples in A were determined by immunoblotting, and are plotted against the replication values shown in B (open squares). Also shown is the 250 mM salt -washed chromatin from Fig 4 (diamond) and interphase chromatin incubated in metaphase extract from Fig 6 (circle).
Figure 2
Figure 2
Removal of XOrc1 by exposure to high salt at different stages of origin assembly. Xenopus sperm nuclei were incubated for 15 minutes in interphase extracts previously immunodepleted with antibodies against XOrc1, XCdc6, or XMcm3 or in undepleted interphase or metaphase extract. Untreated sperm nuclei were used as control. Chromatin was isolated through Nuclear Isolation Buffer containing either 50 mM KCl (“L”) or 200 mM KCl (“H”), and was then immunoblotted for XOrc1, XCdc6 or XMcm3.
Figure 3
Figure 3
Activity of chromatin-bound XORC during assembly of licensed origins. A. Xenopus sperm nuclei were incubated for 15 minutes in interphase Xenopus extracts previously immunodepleted with antibodies against XOrc1, XCdc6, XMcm3 or with non-immune antibodies, or in control extract. Chromatin was isolated and then re-incubated for a further 15 minutes in a second aliquot of XOrc1-depleted extract, before being transferred to 6-DMAP treated extract and incubated for a further 90 minutes in the presence of [α-32P]dATP, to assess the degree of licensing. In order to control for varying chromatin recoveries, the extent of licensing was expressed as the percentage of that seen when the template was licensed in control extract. B, C. Chromatin was prepared by incubating Xenopus sperm nuclei for 15 minutes either in control extract (for interphase chromatin,) or in 6-DMAP-treated extract (for 6-DMAP chromatin), and was then washed in Nuclear Isolation Buffer containing either 50 mM KCl or 250 mM KCl. Aliquots of chromatin were either (B) incubated in untreated interphase extract or 6-DMAP treated extract and the extent of replication over 90 minutes measured, or (C) immunoblotted for the presence of XOrc1 and XOrc2.
Figure 4
Figure 4
Removal of XORC and XCdc6 by high-salt treatment. Xenopus sperm nuclei were incubated for 15 minutes in untreated interphase Xenopus extract. Chromatin was then isolated in buffers containing increasing concentrations of KCl. A. Chromatin was immunoblotted for XOrc1, XOrc2, XCdc6 and XMcm3. Untreated Xenopus sperm were also blotted as a control. B. Chromatin was transferred to XOrc1-depleted extract. Untreated sperm nuclei were used as control. Following incubation in the presence of [α-32P]dATP for 140 minutes, the total amount of DNA synthesised was determined. DNA synthesis is expressed as a percentage of that obtained in extract immunodepleted using non-immune antibodies.
Figure 5
Figure 5
Replication of high salt-washed chromatin in XOrc1- and XCdc6-depleted extracts is sensitive to p21Cip1. Xenopus sperm nuclei were incubated for 15 minutes in untreated interphase Xenopus extract, and chromatin was then isolated in Nuclear Isolation Buffer containing 250 mM KCl. This high salt-washed chromatin, or untreated sperm nuclei, were incubated for 140 minutes with [α-32P]dATP plus or minus p21Cip1 in extract immunodepleted with antibodies to A, XOrc1 or B, XCdc6. DNA synthesis is expressed as a percentage of that obtained in extract immunodepleted using non-immune antibodies.
Figure 6
Figure 6
Removal of XOrc1 by exposure to metaphase extract at different stages of origin assembly. Xenopus sperm nuclei were incubated for 15 minutes in interphase extracts previously immunodepleted with antibodies against XOrc1, XCdc6, or XMcm3 or in control interphase extract. Untreated sperm nuclei were used as control. Incubations were divided into two equal aliquots, and one aliquot was supplemented with 2 volumes of metaphase extract (“+ M-extract”) for a further 15 minutes. A, B. Chromatin was then isolated and immunoblotted for XOrc1, XOrc2, XCdc6 or XMcm3 as indicated. C, The chromatin samples shown in panel B were incubated for 140 minutes with [α-32P]dATP in XOrc1-depleted extract or in extract treated with 6-DMAP, after which the total amount of DNA synthesised was determined. DNA synthesis is expressed as a percentage of that obtained in extract immunodepleted with non-immune antibodies.
Figure 7
Figure 7
Proposed steps in the assembly of licensed origins. A short section of DNA is shown at different stages during the assembly of a functional licensed origin. A. In metaphase, XORC binds only weakly to chromatin and can be eluted with 200 mM salt. B. On exit from metaphase, XORC binding to chromatin becomes resistant to salt elution. C. XCdc6 then associates with the origin, possibly by binding directly to XORC. D. Licensing of the origin by RLF-B and RLF-M results in multiple copies of RLF-M being assembled onto the chromatin at sites distinct from XORC and XCdc6. E. As a consequence of licensing, the binding of XORC is destabilised so it can be removed by exposure to high salt or high Cdk levels. Once licensing has occurred, XORC and XCdc6 have fulfilled their essential functions in DNA replication, and can be removed from the chromatin without comprising the licensed state of the origin.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bell SP, Stillman B. ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex. Nature. 1992;357:128–134. - PubMed
    1. Blow JJ. Preventing re-replication of DNA in a single cell cycle: evidence for a replication licensing factor. J. Cell Biol. 1993;122:993–1002. - PMC - PubMed
    1. Blow JJ, Laskey RA. Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs. Cell. 1986;47:577–587. - PubMed
    1. Blow JJ, Laskey RA. A role for the nuclear envelope in controlling DNA replication within the cell cycle. Nature. 1988;332:546–548. - PubMed
    1. Blow JJ, Nurse P. A cdc2-like protein is involved in the initiation of DNA replication in Xenopus egg extracts. Cell. 1990;62:855–862. - PubMed

Publication types