Cell cycle regulation of the replication licensing system: involvement of a Cdk-dependent inhibitor

Abstract

The replication licensing factor (RLF) is an essential initiation factor that is involved in preventing re-replication of chromosomal DNA in a single cell cycle. In Xenopus egg extracts, it can be separated into two components: RLF-M, a complex of MCM/P1 polypeptides, and RLF-B, which is currently unpurified. In this paper we investigate variations in RLF activity throughout the cell cycle. Total RLF activity is low in metaphase, due to a lack of RLF-B activity and the presence of an RLF inhibitor. RLF-B is rapidly activated on exit from metaphase, and then declines during interphase. The RLF inhibitor present in metaphase extracts is dependent on the activity of cyclin-dependent kinases (Cdks). Affinity depletion of Cdks from metaphase extracts removed the RLF inhibitor, while Cdc2/cyclin B directly inhibited RLF activity. In metaphase extracts treated with the protein kinase inhibitor 6-dimethylaminopurine (6-DMAP), both cyclin B and the RLF inhibitor were stabilized although the extracts morphologically entered interphase. These results are consistent with studies in other organisms that invoke a key role for Cdks in preventing re-replication of DNA in a single cell cycle.

Figure 4

Evidence for a Cdk-dependent RLF inhibitor. (a) Metaphase-arrested extracts were depleted of Cdks with an equal volume of p13^suc1-coupled Sepharose. Depleted extract was subjected to serial dilution, and then mixed with an equal volume of either buffer (○̶ ), crude RLF-B (●̶ ), crude RLF-M (□̶ ), or a mixture of crude RLF-B and crude RLF-M (▲̶ ). B, buffer in place of diluted extract. Unlicensed chromatin was incubated in the mixture (12 ng DNA/μl) for 15 min at 23°C to allow licensing, and then was transferred to 2.5 vol 6-DMAP–treated extract containing [α³²P]dATP. The total DNA synthesized after further incubation for 90 min at 23°C was measured. (b) Preincubations were performed for 15 min with different combinations of partially purified RLF-B, purified RLF-M, and Cdc2/cyclin B (MPF). Unlicensed chromatin was then added, and the incubation was continued for an additional 15 min to allow licensing to occur. Samples were then transferred to 6-DMAP–treated extract containing [α³²P]dATP, and the total DNA synthesized after further incubation for 90 min at 23°C was measured.

Figure 1

Effect of serial dilution on XMcm3 assembled onto chromatin and rate of replication. Interphase Xenopus extract was subjected to serial dilution, and 30-μl aliquots (or 60-μl for a, lanes 3 and 10) were incubated with equal quantities (360 ng DNA) of unlicensed chromatin (a, lanes 3–9; b) or sperm nuclei (a, lanes 10–16) for 15 min at 23°C. (a) Equal quantities of chromatin (∼300 ng DNA) were isolated by centrifugation through sucrose, run on a 7.5% polyacrylamide gel, and immunoblotted with an anti-XMcm3 antibody. Dilutions were: (lanes 3 and 10) undiluted (60 μl); (lanes 4 and 11) undiluted (30 μl); (lanes 5 and 12) 1/2; (lanes 6 and 13) 1/4; (lanes 7 and 14) 1/8; (lanes 8 and 15) 1/16; (lanes 9 and 16) 1/32. Samples of recombinant GST-tagged XMcm3 (lane 1, 40 ng; lane 2, 20 ng) and interphase Xenopus egg extract (lane 17, 0.125 μl; lane 18, 0.25 μl; lane 19, 0.5 μl) were blotted in parallel. (b) Samples were transferred to 6-DMAP– treated extract containing [α³²P]dATP. The total DNA synthesized at different times during an incubation at 23°C was measured. Samples corresponding to the blots in a are indicated with identical symbols.

Figure 2

RLF activity in serial dilutions of metaphase and interphase extract. Metaphase arrested (a) or interphase (b) Xenopus extracts were subjected to serial dilution, and then mixed with an equal volume of either buffer (○̶ ), crude RLF-B (●̶ ), crude RLF-M (□̶ ), or a mixture of crude RLF-B and crude RLF-M (▲̶ ). B, buffer in place of diluted extract. Chromatin was incubated in the mixture (12 ng DNA/μl) for 15 min at 23°C to allow licensing, and then was transferred to 2.5 vol 6-DMAP–treated extract containing [α³²P]dATP. The total DNA synthesized after further incubation for 90 min at 23°C was measured.

Figure 3

Time course of RLF activity during the in vitro cell cycle. Interphase extract (b) or metaphase extract released into interphase with CaCl₂ (a) was incubated at 23°C for various times. Extract was then diluted eightfold and mixed with an equal volume of either buffer (○̶ ), crude RLF-B (●̶ ), or crude RLF-M (□̶ ). Unlicensed chromatin was incubated in the mixture (12 ng DNA/μl) for 15 min at 23°C to allow licensing, and then was transferred to 2.5 vol 6-DMAP–treated extract containing [α³²P]dATP. The total DNA synthesized after further incubation for 90 min at 23°C was measured. M, metaphase-arrested extract that was not released with CaCl₂.

Figure 5

Features of RLF-M prepared from metaphase or interphase extract. (a) Unlicensed chromatin was incubated for 15 min at 23°C with interphase extract, metaphase extract, or mixtures of RLF-B and RLF-M^meta (RLF-M prepared from metaphase extract) and RLF-M^inter (RLF-M prepared from interphase extract). Samples were then transferred to 6-DMAP–treated extract containing [α³²P]dATP. The total DNA synthesized after further incubation for 90 min at 23°C was measured. (b) Protein samples electrophoresed on a 7.5% polyacrylamide gel and immunoblotted with an anti-XMcm4 antibody. Samples: 1, metaphase extract; 2, RLFM^meta; 3, interphase extract; 4, RLF-M^inter; 5, metaphase extract depleted with p13^suc1-Sepharose; 6, metaphase extract treated with 3 mM 6-DMAP before CaCl₂ release (6-DMAP–treated extract); 7, metaphase extract treated with 2 μM okadaic acid before CaCl₂ release; 8, metaphase extract diluted eightfold and incubated for 15 min at 23°C; 9, metaphase extract diluted eightfold, mixed with crude RLF-B, and incubated for 15 min at 23°C.

Figure 6

Effect of cycloheximide and Cip1 of RLF stability. (a) Metaphase extract was released into interphase with CaCl₂ either untreated (control) or in the presence of 100 μg/ml cycloheximide (+ CHX), or 100 μg/ml cycloheximide plus 150 nM p21^Cip1 (+ CHX + Cip1). At the indicated times, samples were diluted eightfold and incubated for 15 min with unlicensed chromatin. Samples were then transferred to 6-DMAP–treated extract containing [α³²P]dATP. The total DNA synthesized after further incubation for 90 min at 23°C was measured. (b) Interphase extract was incubated at 23°C for 2 h to allow RLF activity to decay. The extract was subjected to serial dilution, and then mixed with an equal volume of either buffer (○̶ ), crude RLF-B (●̶ ), crude RLF-M (□̶ ), or a mixture of crude RLF-B and crude RLF-M (). B, buffer in place of diluted extract. Unlicensed chromatin was incubated in the mixture (12 ng DNA/μl) for 15 min at 23°C to allow licensing, and then was transferred to 2.5 vol 6-DMAP–treated extract containing [α³²P]dATP. The total DNA synthesized after further incubation for 90 min at 23°C was measured.

Figure 7

Analysis of the stage of replication blocked in 6-DMAP– treated extracts. (a) Alkaline agarose gel of nascent DNA. (Lanes 1–4) Chromatin was licensed for 15 min with either buffer (lane 1), RLF-B (lane 2), RLF-M (lane 3), or RLF-B plus RLF-M (lane 4), transferred to 6-DMAP–treated extract containing [α³²P]dATP, and incubated for 90 min. (Lanes 5–9) Sperm nuclei was incubated for 90 min in interphase Xenopus extract containing [α³²P]dATP and various concentrations of aphidicolin (lane 5, 30 μg/ml; lane 6, 20 μg/ml; lane 7, 10 μg/ml; lane 8, 5 μg/ml; lane 9, no aphidicolin). To compensate for the much higher incorporation of ³²P in the reactions, the total DNA loaded in lane 4 was reduced to 20%, and the total DNA in lane 9 was reduced to 10%. (b and c) BrdUTP density substitution of nascent DNA synthesized after incubation of sperm nuclei for 90 min in untreated (c) or 6-DMAP–treated extract (b).

Figure 8

Effect of 6-DMAP on RLF activity. (a) Unlicensed chromatin was incubated for 15 min plus or minus 3 mM 6-DMAP with crude RLF-B, crude RLF-M, or a mixture of the two. Samples were then transferred to 6-DMAP–treated extract containing [α³²P]dATP. The total DNA synthesized after further incubation for 90 min at 23°C was measured. (b) Metaphase-arrested Xenopus extract was treated with 3 mM 6-DMAP and 0.3 mM CaCl₂, followed by serial dilution in buffer containing 3 mM 6-DMAP. Samples were then mixed with equal volumes of either buffer (○̶ ), crude RLF-B (●̶ ), crude RLF-M (□̶ ), or a mixture of crude RLF-B and crude RLF-M (▲̶ ). B, buffer in place of diluted extract. Chromatin was incubated in the mixture (12 ng DNA/μl) for 15 min at 23°C to allow licensing, and then was transferred to 2.5 vol 6-DMAP–treated extract containing [α³²P]dATP. The total DNA synthesized after further incubation for 90 min at 23°C was measured.

Figure 9

Titration of 6-DMAP into metaphase extract. (a) Metaphase extract was labeled for 20 min with [³⁵S]methionine, and then was supplemented with 100 μg/ml cycloheximide and the indicated concentrations of 6-DMAP. Extract was then further supplemented plus (lanes 2–8) or minus (lane 1) 0.3 mM CaCl₂. After incubation at 23°C for 30 min, cyclin–Cdk complexes were collected on p13^suc1 beads, electrophoresed on polyacrylamide gels, and autoradiographed. The migration of cyclin B is indicated. (b) Metaphase extract was supplemented with sperm nuclei (3 ng DNA/μl) and various concentrations of 6-DMAP, plus (lower panel) or minus (upper panel) 0.3 mM CaCl₂. The morphology of chromatin after 1.5 h at 23°C was assessed by microscopy: (□), condensed chromosomes; (▪), interphase nuclei; (▨ ), partial nuclear assembly. (c) Metaphase extract was supplemented with sperm nuclei (3 ng DNA/μl), 0.3 mM CaCl₂, [α³²P]dATP, and various concentrations of 6-DMAP. The total DNA synthesized after 3 h at 23°C was measured.