Stepwise assembly of initiation proteins at budding yeast replication origins in vitro

Abstract

The initiation of DNA replication in the budding yeast Saccharomyces cerevisiae occurs in two sequential and mutually exclusive steps. Prereplicative complexes (pre-RCs) containing origin recognition complex (ORC), Cdc6p, and the MCM2-7 proteins assemble only under conditions of low cyclin-dependent kinase (Cdk) activity during G(1), whereas origin activation is driven by the increase in Cdk activity at the end of G(1). As a first step toward the reconstitution of this two-step process in vitro, we describe a system in which extracts prepared from G(1)-arrested cells promote sequential assembly of ORC, Cdc6p, and MCM2-7 proteins onto exogenously added origin-containing DNA. This reaction requires an intact ARS consensus sequence and requires ATP for two distinct steps. Extracts from cells arrested in mitosis also can support the binding of ORC but are unable to load either Cdc6p or MCM2-7 proteins. This system should be useful for studying the mechanism and regulation of pre-RC assembly.

Figure 1

A cell-free system for the assembly of pre-RCs. (A–C) Preparation of ARS1 beads. (A) Complementary oligonucleotides for ARS1 beads. The positions of the A, B1, and B2 elements are indicated by open boxes. In the mutated ARS1 oligo DNA, 8 bp of the A element was replaced with an 8-bp XhoI linker sequence. (B) Preparation of ARS1 beads. See Materials and Methods for details. (C) Aliquots of annealed wild-type (W) and mutant (M) oligonucleotides, before (1 and 3) and after (2 and 4) the ligation, were run on an agarose gel and stained with ethidium bromide. (D–F) Assembly of pre-RC components on ARS1 beads. Whole-cell extracts prepared from YGP82 cells expressing Cdc6p were incubated with either wild-type (W) or mutant (M) ARS1 beads. (D) ORC loading. (E) Cdc6p loading. (F) Mcm loading. To examine the loading of Mcm7p, whole-cell extracts were prepared from YCD2 cells, and Mcm7p tagged with c-myc epitope was detected with the 9E10 antibody. (G) The levels of pre-RC components in reaction mixtures. After the incubation, samples were taken from the supernatants separated from the wild-type (ARS1 W) or mutant (ARS1 M) beads. A reaction mixture lacking ARS1 beads was constructed separately, and a sample was taken without an incubation (ARS1 −). The levels of pre-RC components were detected by immunoblotting (Sup).

Figure 2

Cdc6p-dependent protein loading on ARS1 beads. Whole-cell extracts were prepared from YGP82 cells in which Cdc6p was expressed (Cdc6p +) or repressed (Cdc6p −). Wild-type (W) or mutant (M) ARS1 beads were incubated with increasing amounts of the extracts, as indicated by their final concentrations in the reaction mixtures (Extracts). The levels of ORC, Cdc6p, and Mcm proteins associated with the beads (Bead) or in the supernatants (Sup) were detected by immunoblotting as described in Fig. 1.

Figure 3

ATP requirement for protein assembly on ARS1 beads. (A) ARS 1 beads were incubated with whole-cell extracts prepared from YGP82 cells expressing Cdc6p either in the presence of (ATP +) or in the absence of (ATP −) exogenously added ATP and the ATP regenerating system. Proteins associating with the beads (Bead) or remaining in the supernatants (Sup) were detected by immunoblotting as before. (B) Loading reactions were carried out either with or without exogenously added ATP and the ATP regenerating system (ATP + or −). To the indicated reactions (Apyrase +), 5 units of apyrase was added. After incubation with ARS1 beads, Orc2p associating with the beads (Bead) and remaining in the supernatants (Sup) was detected.

Figure 4

Pre-RC assembly can occur in extracts of G₁ cells but is blocked in extracts of G₂/M cells. Whole-cell extracts prepared from Cdc6p-expressing cells (YGP82), arrested either in G₁ phase with α-factor (α) or in G₂/M with nocodazole (Noc), were incubated with wild-type (W) or mutant (M) ARS1 beads. The levels of ORC, Cdc6p, and Mcm2p associated with the beads (Bead) or present in the supernatants (Sup) were determined as above.