In the experiment studying ORC-DNA interaction in Xenopus egg extract, described
in Chapter 8, Figure 8/4, 500 |il of interphase extract was suplemented with 25 mM
phosphocreatine, 10 pg/ml creatine phosphokinase and 2 mM MgClj. 12.5 )ig of
pARSl-1.4.1 plasmid was then added and the sample was incubated for 15 min at 23°C and spun at 14 000 rpm for 8 min at 4°C in a bench top centrifuge. 200 |il of obtained supernatant was then incubated in the presence of 150 pi o f binding buffer (50 mM Tris, pH 7.5; 150 mM NaCl; 0.1% NP-40; 1 mM DDT) with 15 pi o f protein A beads coupled to either anti-Orclp antibody T K l5 or to a control, anti- SV40 T-antigen antibody, and rotated for 2 hours at 4°C. The beads were then washed three times with the binding buffer (50 mM Tris pH 7.5; 150 mM NaCl;
C hapter 2
examine whether ORC was bound to the beads. The remaining beads were incubated with 150 III of 50 mM Tris pH 8.0/10 mM EDTA/1% SDS at 65"C for 20 min. Then 140 |il o f TE (10 mM Tris pH 8.0; 1 mM EDTA), 2 |il o f glycogen (20 mg/ml, Boehringer Mannheim) and 7.5 jiil of proteinase K (20 mg/ml) were added and the samples were incubated for 2 hours at 37°C. Samples were then extracted with phenol and chloroform, precipitated with ethanol, resuspended in TE (10 mM Tris pH8.0; 1 mM EDTA), treated with RNAse and analysed by agarose gel. electrophoresis.
ORC-DNA binding in vitro
Figure 8/5 in Chapter 8 shows an experiment in which 48 |il of protein A beads coupled to anti-Orclp antibody T K l5 were incubated with 800 |il of interphase extract in the presence of 400 jj.1 of binding buffer (36 mM Hepes, pH 7.6; 0.72 mM EDTA; 0.72 mM EGTA; 3.6 mM Magnesium acetate; 7.2% glycerol; 120 mM KCl; 0.2% NP-40) for 1 hour rotating at 4°C. Beads were then washed three times with the binding buffer (36 mM Hepes, pH 7.6; 0.72 mM EDTA; 0.72 mM EGTA; 3.6 mM Magnesium acetate; 7.2% glycerol; 120 mM KCl; 0.2% NP-40) and 6 |xl o f beads were used per binding reaction. Binding reactions were carried out in 50 jiil final volume in the binding buffer (36 mM Hepes, pH 7.6; 0.72 mM EDTA; 0.72 mM EGTA; 3.6 mM Magnesium acetate; 7.2% glycerol; 120 mM KCl; 0.2% NP-40). To each reaction, 2 jug of DNA fragments produced by a restriction digest o f pA R Sl- 1.4.1 plasmid, and end labelled with [y-^^P] ATP using T4 polynucleotide kinase and subsequent purification on G 50 Sepharose coloum, were added. Reactions were performed either in the presence or in the absence of 1 mM ATP, 10 mM phosphocreatine and 4 gg/ml creatine phosphokinase. Increasing amounts o f DNA competitor poly (dl-dC) was added to the reactions to final concentrations o f 0, 2 and
6 jig/ml. Binding reactions were carried out by rotating for 15 min at room temperature and samples were washed three times with binding buffer (36 mM Hepes, pH 7.6; 0.72 mM EDTA; 0.72 mM EGTA; 3.6 mM Magnesium acetate; 7.2% glycerol; 120 mM KCl; 0.2% NP-40) following the binding. Bound DNA was
extracted by incubating the beads with 150 \i\ of 50 mM Tris pH 8.0; 10 mM EDTA;
1% SDS at 65°C for 20 min. Then 140 |xl of TE (10 mM Tris pHS.O; 1 mM EDTA),
2 |xl o f glycogen (20 mg/ml, Boehringer Mannheim) and 7.5 |Lil o f proteinase K
(20 mg/ml) were added and the samples were incubated for 2 hours at 37°C. Samples were then extracted with phenol and chloroform, precipitated with ethanol, resuspended in TE (10 mM Tris, pH 8.0; 1 mM EDTA) and analysed on a 5% non denaturing acrylamide gel.
Chapter 3
Immunopurification of Xenopus ORC
I started my PhD shortly after the Xenopus Ore Ip was identified as a result of
collaboration of three ICRF labs (Rowles et aL, 1996). The idea behind my project
was to re-address the controversial question o f specificity of replication origins in higher eukaryotes (see Introduction). The strategy was to attempt the identification
o f DNA sequenced X enopus ORC recognises. I used the Ore Ip subunit o f the
presumed X en opus ORC to get a handle on the whole complex. This chapter
describes raising a number o f monoclonal antibodies against the Ore Ip protein,
development of Xenopus ORC immunopurification scheme and isolation of pure and
presumably complete Xenopus ORC.
Polyclonal antisera to Xenopus Ore Ip an Orc2p
I first generated polyclonal antibodies against Xenopus Ore Ip in rabbits. These were
useful to assess the results of immunoaffinity purification of ORC using anti-Orclp monoclonal antibodies raised in mice. Mouse antibodies used for immunoaffinity purification can be present in the sample of the eluted protein. When such sample is analysed by immunobloting with anti-mouse secondary antibody, the mouse antibody
on the blot is recognised, giving strong interfering signal. Using anti-rabbit
To generate polyclonal antibodies against the Xenopus Ore Ip the full length open
reading frame was subcloned into pET21b vector so as to add a hexahistidine tag to the C-terminus (see Methods). This fusion protein was expressed very efficiently in bacteria (see Methods). The majority of the produced protein was insoluble and was solubilised in guanidine hydrochloride prior to the purification on nickel agarose beads (see Methods). Figure 3/1 shows that the purification yielded a preparation of relatively pure polypeptide of molecular weight of approximately 110 kDa as
expected for full length Xenopus Ore Ip protein. Two rabbits (TK2 and TK3) were
injected with 7 x 500 pg doses of the purified Ore Ip protein. Figure 3/2 shows that
the preimmune sera did not recognise any protein in the Xenopus egg extract. The
immune respose increased with the increasing number o f immunisations. Antisera (final bleed) produced by both rabbits recognised a band o f the molecular weight corresponding to Ore Ip on an immunoblot of frog egg extracts. The cross-reacting band was o f very different molecular weight than Ore Ip and therefore did not interfere with the use of these antibodies for inununoblotting. The TK3 antibody was used for all subsequent experiments, as it produced cleaner immunoblot.
The cloning o f Xenopus Orc2p by Phil Carpenter allowed generation of antibodies
against another subunit of frog ORC complex (Carpenter et al., 1996). These
antibodies became very important for determining whether the Orc2p protein co purified with the Ore Ip. This, in turn, served as an indicator, whether the whole ORC complex remained intact during the ORC purification procedure.
The clone encoding Xenopus Orc2p was kindly given to me by Phil Carpenter (see
C hapter 3