Site-specific DNA binding of the Schizosaccharomyces pombe origin recognition complex is determined by the Orc4 subunit

Abstract

The mechanism by which origin recognition complexes (ORCs) identify replication origins was investigated using purified Orc proteins from Schizosaccharomyces pombe. Orc4p alone bound tightly and specifically to several sites within S. pombe replication origins that are genetically required for origin activity. These sites consisted of clusters of A or T residues on one strand but were devoid of either alternating A and T residues or GC-rich sequences. Addition of a complex consisting of Orc1, -2, -3, -5, and -6 proteins (ORC-5) altered neither Orc4p binding to origin DNA nor Orc4p protection of specific sequences. ORC-5 alone bound weakly and nonspecifically to DNA; strong binding required the presence of Orc4p. Under these conditions, all six subunits remained bound to chromatin isolated from each phase of the cell division cycle. These results reveal that the S. pombe ORC binds to multiple, specific sites within replication origins and that site selection, at least in vitro, is determined solely by the Orc4p subunit.

FIG. 1

Purified S. pombe Orc4 protein and ORC-5 complex. Purified Orc4p and purified ORC-5 complex were fractionated by electrophoresis in SDS–8% polyacrylamide gels. Proteins in the Orc4p gel were stained with Coomassie blue, and those in the ORC-5 gel were stained with silver nitrate. Based on the migration of standard proteins (Bio-Rad) fractionated in parallel on the same gel, molecular masses were estimated as Orc1p (95 kDa), Orc2p (72 kDa), Orc3p (79 kDa), Orc4p (130 kDa), Orc5p (48 kDa), and Orc6p (29 kDa).

FIG. 2

Orc4p binds specifically to S. pombe DNA replication origins. (A) DNA band shift assays were carried out by incubating the indicated amount of Orc4p with either ³²P-labeled S. pombe replication origin ARS3002 (363 bp) or ARS1 (853 bp) or with a control DNA (688 bp) consisting of an average sequence. Samples were then fractionated by agarose gel electrophoresis, and [³²P]DNA-Orc4p complexes were detected by autoradiography. (B) Competition between ARS3002 [³²P]DNA (5 ng) and either unlabeled ARS3002 DNA or control DNA was detected by incubating the indicated molar ratio of DNA with 2 ng of Orc4p. Molar DNA ratios were that of unlabeled competitor DNA to [³²P]DNA. Conditions were the same as for panel A.

FIG. 3

ORC-5 selectively binds to Orc4p-DNA complexes. DNA band shift assays using [³²P]ARS3002 were carried out as for Fig. 3. The amounts of Orc4p, ORC-5, and ATP added to each reaction are indicated. In lanes 4, 5, 8, and 9, Orc4p was allowed to bind to DNA for 5 min before addition of ORC-5. In lanes 7 and 11, Orc4p and ORC-5 were combined, kept on ice for 5 min, and then added to the reaction.

FIG. 4

The ORC-5 complex binds to the Orc4p-origin DNA complex. An aliquot of the ORC-5 complex (lane 1) was passed through ARS3002 cellulose (lane 2) or ARS3002-Orc4p cellulose (lane 3) to which an aliquot of Orc4p (lane 1) had been prebound (lane 4). The column was washed with starting buffer (buffer A which contains 0.1 M KCl) to remove unbound proteins and then with 1 M KCl (buffer A + 0.9 M KCl) to elute bound ORC proteins. ORC proteins in the eluent were fractionated by electrophoresis in an SDS-polyacrylamide gel and were then detected by immunoblotting using antisera against Orc1, -2, -4, and -6 proteins.

FIG. 5

All six ORC subunits are bound to chromatin in 100 mM KCl. To get chromatin from different phases of cells, S. pombe strains JL197 ura4-D18 cdc10-129 (G₁), JL202 ura4-D18 cdc22-M45 (S), and JL206 ura4-D18 cdc25-22 (G₂) were grown to an OD₅₉₅ of 0.5 at 26°C and were then shifted to 36.5°C for an additional 3.5 h. To arrest cells at the M phase, 25 μg of benomyl/ml was added to a cell culture with an OD₅₉₅ of 0.5, and the culture was incubated for an additional 3.5 h. Chromatin was prepared and analyzed as described in Materials and Methods.

FIG. 6

Orc4 protein binds to specific sites in ARS3002. A 363-bp DNA fragment containing S. pombe ARS3002, labeled at one of its 5′ ends, was incubated with the indicated amount of Orc4p and then digested with DNase I (0.2 U for bare DNA, 0.5 U for 10 ng of Orc4p, and 2 U for 40 to 80 ng of Orc4p) as previously described (25). The DNA binding conditions for Orc4p and ORC-5 complex were the same as described for DNA band shift experiments. Lanes G, A, T, and C refer to DNA sequencing obtained using the dideoxyribonucleotide method according to the manufacturer's instructions. A DNA primer 25 nucleotides long was annealed to the 3′ end of the strand that was complementary to the 5′ ³²P-labeled strand in order to reveal the sequence of the 5′ ³²P-labeled strand. Sequences protected by Orc4p are indicated as A, B, and C.

FIG. 7

The ORC-5 complex neither binds to specific sites of its own nor alters the binding pattern of Orc4p. The experiment described for Fig. 6 (Watson strand) was carried out in the presence of the indicated amounts of Orc4p, ORC-5 complex, and ATP using the amounts of DNase I indicated. When both Orc4p and ORC-5 were used, they were mixed together before addition of [³²P]DNA. Protected regions A, B, and C are indicated.

FIG. 8

The 363-bp sequence for ARS3002 (49) was used for DNA band shift assays and for in vitro footprinting experiments. Regions A, B, and C, protected by Orc4p, are in boldfaced italics. Two regions required for ARS3002 activity (deletions 8 and 10 [49]) are doubly underlined. One region not required for ARS3002 activity (deletion 6) is wavily underlined. This region contains a block of alternating AT residues.

FIG. 9

Site-specific DNA binding by the S. pombe ORC is determined solely by the Orc4p subunit, which directs ORC to several specific sites (rectangular boxes in corners) within S. pombe DNA replication origins. In vitro, purified ORC-5 complex did not bind to replication origins in the absence of purified Orc4p, but in vivo, Orc4p and ORC-5 may exist as a single protein complex that binds to newly replicated origins in a single step.