Inhibition of DNA replication by geminin is mediated by inhibition of RLF-B.
a, Unlicensed chromatin isolated from 6-DMAP-treated extract (“6-DMAP chromatin”) was incubated in partially-purified licensing factor fractions (MQE and BPAS). 35 nM recombinant geminin was added either before or after incubation with licensing factor fractions, or not at all. After incubation for 20 mins, the extent of licensing was assayed by addition of 6-DMAP-treated extract containing [α-³²P]dATP and incubation for a further 90 mins. b, Fractionation scheme for origin components from Xenopus extracts. c, d, Interphase egg extract was treated with geminin-coupled agarose beads or with control beads, and was supplemented with various fractions. After incubation for 20 mins with sperm nuclei, the extent of licensing was assayed. Inset: BPAS (1), BQE (2), and BQFT (3) immunoblotted for Orc1 and Cdc6.

Purification of RLF-B.
a, Geminin-coupled agarose beads were incubated with BPAS, washed with different buffers, and RLF-B was eluted with buffer containing 4 M urea. Fractions were desalted and assayed for RLF-B activity. >65% of the applied RLF-B activity was recovered in the eluate. Dashed line: buffer alone control. Inset: BPAS (1), geminin bead flow-through (2), and 4 M urea eluate (3) immunoblotted for Orc1, Cdc6 and Cdt1. b, Geminin bead eluate (fractions 33-37) were fractionated by Superose 6 gel filtration and assayed for RLF-B activity. Molecular weight markers (in kDa) are above. Dashed line, buffer alone control. c, 0.25 ml aliquots from the Superose 6 column were precipitated with deoxycholate/TCA and run on a 10% SDS acrylamide gel. Top panel: silver stain of the gel. Molecular weight markers (kDa) are to the left. Arrowhead: ~75 kDa band co-eluting with RLF-B. Small arrows: contaminating keratin bands. Bottom panel: fractions immunoblotted for Cdt1.

Recombinant Cdt1 can provide RLF-B activity.
a, Unlicensed “6-DMAP chromatin” was incubated in interphase extract, a crude fraction containing Mcm(2-7) activity (RLF-M) and 10 nM recombinant Cdt1. After 20 mins, the extent of licensing was assayed by addition of 6-DMAP-treated extract containing [α-³²P]dATP and incubation for a further 90 mins. b, Unlicensed “6-DMAP chromatin” was incubated in interphase extract depleted with either non-immune antibody, anti-Cdt1 antibody, or with recombinant geminin beads, plus or minus purified RLF-B. After 20 mins, the extent of licensing was assayed by addition of 6-DMAP-treated extract containing [α-³²P]dATP and incubation for a further 90 mins. c, d, Interphase extract containing [α-³²P]dATP and sperm nuclei was supplemented with 35 nM recombinant geminin, 20 nM recombinant Cdt1 or 0.25 vols purified RLF-B. After 90 mins, total DNA synthesis was measured.

RLF-B/Cdt1 can associate with geminin in interphase extracts.
a, Interphase extract was supplemented with 35 nM recombinant geminin and then immunodepleted with either non-immune (1) or anti-geminin (2) antibodies. Inset, samples were blotted for either Cdt1 or geminin. Unlicensed “6-DMAP chromatin” was incubated in these depleted extracts or interphase extract or the BPAS fraction, plus or minus a crude fraction containing Mcm(2-7) activity (RLF-M). After 20 mins, the extent of licensing was assayed by addition of 6-DMAP-treated extract containing [α-³²P]dATP and incubation for a further 90 mins. b, The BPAS fraction containing partially-purified RLF-B/Cdt1 was fractionated on phenyl Sepharose. Fractions were assayed for RLF-B, for the ability to rescue licensing in Cdt1-depleted extracts, or were immunoblotted for Cdt1 and geminin.

Proteins associated with chromatin in different extracts.
Sperm nuclei were incubated for 20 mins in either metaphase extract, interphase extract, in metaphase extract supplemented with 0.5 or 3 mM 6-DMAP and 0.3mM CaCl₂ (“metaphase+6-DMAP”), or in interphase extract supplemented with 7 or 35 nM geminin. Chromatin was isolated and immunoblotted for Orc1, Cdc6, Cdt1, Mcm7 and geminin.

Characterization of the licensing inhibitor in metaphase extracts
a, Metaphase-arrested extract was mixed with either geminin bead-purified RLF-B or interphase extract. The mixture was incubated for 20 min with “6-DMAP chromatin”, and licensing subsequently assessed. b, Metaphase extract was immunodepleted with anti-geminin antibodies (filled triangles) or non-immune antibodies (open triangles). Serial dilutions in LFB2/50 buffer of depleted extract or untreated metaphase extract (open circles) were then assayed for their ability to inhibit licensing of “6-DMAP chromatin” by an equal volume of interphase extract. Inset: extract depleted with control antibodies (lane 1) or anti-geminin antibodies (lane 2) immunoblotted for geminin. c, Histone H1 kinase activity of interphase extract (1), metaphase extract (2), geminin-depleted metaphase extract (3) or control-depleted metaphase extract (4). d, Morphology of sperm nuclei incubated in metaphase extract (i, ii), geminin-depleted metaphase extract (iii, iv), or interphase extract (v, vi), stained with Hoechst 33258 and viewed under UV fluorescence (i, iii, v) or phase contrast optics (ii, iv, vi). Scale bar 10 μm. e, The indicated dilution of depleted metaphase extract, or untreated metaphase or interphase extract was incubated for 20 min with “6-DMAP chromatin” and licensing subsequently assessed.

Licensing of sperm nuclei in geminin-depleted metaphase extract.
a, The indicated dilution of geminin-depleted metaphase extract (filled triangles), control-depleted metaphase extract (open triangles), or untreated interphase extract (filled circles) was incubated for 20 min with Xenopus sperm nuclei and licensing subsequently assessed. Inset: sperm chromatin incubated for 20 mins in 0.5× control-depleted metaphase extract (lane 1), geminin-depleted metaphase extract (lane 2), or interphase extract (lane 3), was isolated and immunoblotted for Orc1, Cdc6 and Mcm3. b, A 0.25 dilution of geminin-depleted metaphase extract plus or minus RLF-B was incubated for 20 min with sperm nuclei and licensing subsequently assessed. c, A 0.25 dilution of geminin-depleted metaphase extract or control-depleted metaphase extract plus or minus 3mM 6-DMAP or 2mM roscovitine was incubated for 20 min with sperm nuclei; the chromatin was then isolated and licensing assessed. Inset: Sperm chromatin incubated in 0.5× non-immune depleted metaphase extract (lanes 1, 2), geminin-depleted metaphase extract (lanes 3, 4) or interphase extract (lanes 5, 6) minus (lanes 1, 3, 5) or plus (lanes 2, 4, 6) 2 mM roscovitine, isolated and immunoblotted for Mcm7.

Model for licensing control during mitosis.
a, In metaphase, RLF-B/Cdt1 is inhibited by geminin. CDKs play a minor role in preventing origin assembly by reducing the affinity of ORC and Cdc6 for replication origins, or possibly by direct inhibition of RLF-B/Cdt1. The Mcm(2-7) heterohexamer (“M”) probably remains active during metaphase. b, On exit from metaphase, geminin and the mitotic cyclins are degraded, thus allowing Mcm(2-7) to be assembled onto origins under the combined action of ORC, Cdc6 and RLF-B/Cdt1. Inhibition of CDK activity in metaphase prevents the degradation of geminin, and so stabilizes the inhibitory state into interphase.