The Loading Assay

2.4.1 Production of ARSl beads

Plasmids based on pUCl 19 were chosen for use in the Loading assay. W ARSl

(pARS/WTA, Marahrens and Stillman, 1992) and A' ARS 1 (pARS/865-872, Marahrens

and Stillman, 1992) are Pucl 19, URA3A, CEN4, ARS 1.4.1 W/A" and are 5822bp in size.

These plasmids were routinely used for the Loading assay. W ARS 1 has an insert with

the A, B l, B2 and B3 regions of A RSl, whereas A" ARSl has the same insert, but with an

Xhol site replacing most (positions 865-872) of the A element (refer to Appendix 1).

In Figure 4 the W and A ARSl plasmids are labelled Large, and compared to a

smaller set of plasmids, in the loading assay. The Small set of plasmids have identical

origin regions to the Large set, W and A A RSl, however do not have the URA3 and

CEN4 sequences and hence, are only 3322bp in size.

Figure 5 compares W and A" plasmid beads to B3" plasmid beads, produced from

the plasmid ABFl (PTS)(Pucl 19, URA3A, CEN4, ARS 1.4.1 756, 758) (Marahrens and

Stillman, 1992). This plasmid contains a double point mutation, (C to G at position 756

and T to C at position 758) in the A bflp binding region, within the B3 element.

All plasmids were prepared using Gigapreps (Qiagen), as per manufacturers

PHOTOPROBE® (S-S) BIOTIN (biotin) and a protocol devised, based on manufacturers

instructions.

Initially the biotin reagent was titrated into a reaction with the plasmid, to

maximise the number of plasmids biotinylated, but minimise the number of biotin groups

per plasmid (see section 3.3 Figure la) and a ratio of 1:18 was chosen for moles of

plasmidibiotin and used in all subsequent reactions.

Routinely reactions were set up with 0.13p,Molar plasmid and 2.34p.Molar biotin

(in 80pil reactions) and incubated under UV 365nM for 30 minutes. Samples were then

recovered and cleaned of excess biotin using two 2-butanol washes. An ethanol

precipitation was performed and the plasmid resuspended in water.

Where it was necessary to determine the proportion of plasmid biotinylated

(Biotin titration. Figure la), samples were incubated with an excess of Dynabeads®M-280

Streptavidin (Dynabeads) (300|Lig Dynabeads/pmole plasmid). In all other cases plasmid-

biotin samples were incubated with 120p,g Dynabeads/pmole plasmid, as determined by a

titration of Dynabeads (see section 3.3, Figure Ic). All incubations were carried out on a

wheel, overnight, at room temperature, in lOmM tris-HCl pH 7.6, ImM EDTA, 2M NaCl,

before washing the DNA-beads on a magnet, with HlOEl (lOmM Hepes-KOH pH 7.6,

ImM EDTA), three times, to remove any unbound plasmid. Washed DNA-beads

(30mg/ml beads) were stored in HlOEl, at 4°C, for up to 2 weeks before use in the

Loading Assay.

Where it was necessary to compare concentrations of DNA in different DNA-bead

samples, samples were heated to 65°C for 20 minutes, with Laemmli buffer, to free bound

xylene cyanol, 30% glycerol in H2O) and run on a 0.8% agarose gel, with 0.5jrg/ml

ethidium bromide, to visualise plasmid recovery (see Figure 1). To estimate the

concentration of plasmid in preparations of beads, liberated plasmid was compared to

samples of known concentration. For the bead preparations of 30mg/ml, described above,

plasmid concentration was estimated at 15nM.

2.4.2 The Loading Assay with extracts

Unless otherwise stated, loading assays were performed in 40pl reactions. A

titration of DNA-beads (Figure 2, section 3.4.1) showed that 300p.g of DNA-beads was

sufficient per reaction (at 3.75nM plasmid, therefore 0.15 pmoles per reaction). The

volume supplied to the reaction by DNA-beads was minimal as once aliquoted, this

component was held in a magnetic separator (available from Dynal) and all supernatant

removed to leave only a bead pellet. 20p,l of extract was supplied per reaction and the

remaining volume made up with a Reaction buffer. The final concentrations of reagents

in each reaction were 24mM hepes pH7.6, 312.5mM sorbitol, lOmM magnesium acetate,

2.5mM EGTA, ImM DTT, 3mM ATP, 20mM creatine phosphate (CP), 20U/ml creatine

phosphate kinase (CPK), 837.5p.g/ml Poly(dl-dC)"Poly(dl-dC)* and the protease

inhibitors: 1 p.g/ml pepstatin A, 10p.g/ml aprotinin, ImM AEBSF, ImM benzamidine,

lOpig/ml leupeptin. There was also a salt concentration equivalent to appoximately

300mM potassium glutamate, provided by the extract (determined by conductivity

DNA-beads were gently resuspended in the reaction mix and tubes placed in an

Eppendorf Thermomixer, for 20 minutes at 24°C (temperature having been optimised in Figure 3.) After the incubation, tubes were recovered on ice then placed in a pre-cooled

(4°C ) magnetic separator, until all beads were settled against the magnet (this takes 2

minutes). Supernatants were then carefully removed holding the pipette tip away from

the bead pellet (and in some cases kept for analysis by immunoblotting). Samples were

then replaced on ice and the DNA-beads gently resuspended in 400p,l of Wash buffer

(50mM hepes pH7.6, 300mM potassium glutamate, 10% w/v glycerol, 5mM magnesium

acetate, ImM EGTA, ImM DTT, 0.1% Triton xlOO, and the protease inhibitors: Ip-g/ml

pepstatin A, 10p,g/ml aprotinin, ImM AEBSF, ImM benzamidine, 10p.g/ml leupeptin) by

pipetting up and down. Resuspended samples were returned to the magnetic separator

and the washing process repeated for a second time. After the removal of all supernatant,

samples were resuspended in 20p.l Laemmli buffer (6.7% w/v glycerol, 0.715mM |3-

mercaptoethanol, 3% SDS, 62.5mM tris-HCl pH 6.9, 0.0042% bromophenol blue) and

boiled for 3 minutes.

*Poly(dI-dC) "Poly(dl-dC) was purchased as double stranded DNA from Amersham

Pharmacia Biotech and dissolved to lOmg/ml in HlOElKOAcSO (lOmM Hepes-KOH

pH7.6, ImM EDTA and 50mM Potassium Acetate), before heating to 45°C for 5 minutes.

The tube was then cooled on ice and sonicated (5 microns amplitude) for 30 seconds,

2.4.3 The Loading Assay with semi-purified ORC

Loading Assays with semi-purified ORC were carried out in the same buffers as

described in 2.4.2. The ORC used was from the elution of a DNA cellulose column and

contained about 300mM KCl (see section 2.5). Reactions were performed at a final

concentration of 60mM KCl and 180mM potassium glutamate. The Loading Assay was

performed at 24°C as described in 2.4.2.

2.4.4 The Loading Assay with competitor DNA

In Figure 6, Loading Assays were carried out as described in 2.4.2. After 20

minutes, when pre-RCs had assembled, W ARSl plasmid was added to be in competition

with W ARSl plasmid bound to Dynabeads. This competitor DNA was at a very high

concentration (25pg/pl) to avoid large dilutions of the reactions and in a control reaction

was replaced by an identical volume of water. ARS 1 plasmid bound to beads was

estimated to be at 4nM (3.75nM). Competitor DNA was added to 40nM (lOx) or 400nM

(lOOx) with an energy stock providing 3mM ATP, 20mM creatine phosphate and 20U/ml

creatine phosphate kinase. Incubations were continued for the times stated, before

isolation of DNA-beads , washing and analysis, as described previously.