9. PRESENTACIÓN Y ANÁLISIS DE LA INFORMACIÓN RECOGIDA
9.2 ESTUDIO DE MERCADOS
9.2.1 Descripción del Servicio
To determine the requirements for ATP and for the ATP regeneration system in
loading assays, a titration of ATP was performed, with and without the ATP regenerating
system. The results are shown in Figure 11a. Seven loading assay reactions were
prepared using ammonium sulphate precipitated and dialysed extracts (to ensure efficient
removal of endogenous nucleotides) and W and A' ARSl plasmid beads. Additions of
regeneration system (20mM creatine phosphate and 20U/ml creatine phosphate kinase).
To the other four tubes (-), 300p.M, ImM, 3mM and lOmM ATP were added, with an
equalizing volume of water. All the reactions were incubated for 20 minutes using the
routine conditions and the bound proteins analyzed by immunob lotting, as described
previously.
Reactions 4 to 7 in Figure 1 la show loading assays performed without the
addition of the ATP regeneration system. On addition of 300[xM of ATP (7) no Orc2p or
Mcm2p was detected in the ARSl bead-bound samples, suggesting that the ATP
concentration was too low to stimulate binding of ORC to origins or for the loading of the
MCM complex. Cdc6p on the other hand, was detected in both the W and A" ARSl
samples at a relatively high concentration. This is consistent with previous experiments,
which have demonstrated that Cdc6p is present in the bead-bound fractions of W and A'
ARSl loading assays when no ATP is added to reactions. We suggest that at low
nucleotide concentrations the conformation o f Cdc6p is affected and that the protein
sticks, nonspecifically, to DNA or to the dynabeads themselves.
When ImM ATP (no regeneration system) was added to reactions (6) Orc2p was
detected in the W ARSl bound fraction, although this was at a relatively low level. Thus,
the addition of ImM ATP stimulated some specific binding of ORC to W ARSl origins.
In these reactions Cdc6p was again detected in both the W and A ARS samples and
Mcm2p was barely detectable, demonstrating that although ORC bound origins, pre-RC
assembly was not stimulated.
When 3mM ATP (no regeneration system) was added to reactions (5) efficient
Figure 11
Titrations of nucleotides/nucleotide analogue
A ATP titrations with and without an ATP regeneration system
Loading Assays were performed with W and A ' ARSl beads and triple ammonium sulphate cut G1 extracts (ammonium sulphate precipitation and dialysis performed three times to maximise removal of nucleotides.) Incubations included ATP concentrations between 300p.M and lOmM, +/- the ATP regenerating system (20mMCP and 20U/mlCPK). After 20 minutes at 24°C, the ARSl beads were isolated and bound proteins analysed by immunoblotting.
Proteins bound to a nitrocellulose membrane were incubated with polyclonals raised against Orc2p and Mcm2p and a monoclonal raised against Cdc6p, using the conditions described in 2.7.3.
B ADP titration
Loading assays were performed with W and A ' ARSl beads and triple ammonium sulphate cut G1 extract, with ADP concentrations between 0 and lOmM (without any ATP regenerating system). A control was also prepared with 3mM ATP (with ATP regenerating system: 20mMCP and 20U/ml CPK) After 20 minutes at 24°C the ARSl beads were isolated and bound proteins analysed by immunoblotting.
Proteins bound to a nitrocellulose membrane were incubated with polyclonals raised against Orc2p and Mcm2p and a monoclonal raised against Cdc6p, using the conditions described in 2.7.3.
C ATPyS titration
IIA
IIB
l i e CP, CPK + A TP 3mM Im M 3 0 0 pM lOmM 3mM Im M 3 0 0 p M A R S l Orc2p W A W A W A W A W A W A W A C dc6p M cm 2p • » • . . . « 1 2 3 4 5 6 7 N ucleotide A TP, CP,CPK A D P 3mM lOmM 3m M ImM 300p M 0 A R S l Orc2p W A W A W A W A W A W A- Orc6p • C dc6p • M cm 2p 1 2 3 4 5 6 N ucleotide A TP, CP,CPK ATPyS 3mM lOmM 3m M ImM 300p M 0 A R S l W A- W A W A W A W A W A O rc2p C dc6p M cm 2preduced and no Mcm2p was detected. These results suggest that this higher concentration
of ATP stimulated more ORC binding to origins and reduced the non-specific sticking of
Cdc6 to DNA/Dynabeads, perhaps by changing the conformation o f Cdc6p. Because
Cdc6p did not bind to W ARS 1 specifically and no Mcm2p loading was detected it seems
that although the conformation of Cdc6p may have been altered by addition of 3mM
ATP, Cdc6p was still not active.
On addition of lOmM ATP (no regeneration system) to reactions (4) Orc2p and
Mcm2p loading specific for W ARSl was detected. Although Cdc6p was detected in
both the W and A' ARS 1 samples, the loading of Mcm2p suggests that some proportion
of Cdc6p was active in respect to the loading of the MCM2-7 complex.
Reactions i to 3 were incubated in the presence of ATP and the ATP regeneration
system. Orc2p bound efficiently to W ARSl on addition o f 300p.M, ImM or 3mM ATP,
suggesting that ATP was in excess, in respect to ORC binding, in all of these reactions.
At 300|liM ATP, Cdc6p was detected in both the W and A' ARSl samples. In the
presence of ImM ATP loading of Cdc6p was more efficient for W ARSl and in the
presence of 3mM ATP Cdc6p was bound only to W A RSl. Thus, Cdc6p loading became
more specific for W ARSl origins at increased concentrations of ATP, suggesting that
higher proportions of Cdc6p were converted to an active conformation. Mcm2p loading
was detected in all three reactions with the ATP regeneration system, and increased in
efficiency with increased ATP concentration.
The loading assays with the ATP regeneration system have therefore
demonstrated that the different components of the pre-RC have different thresholds of
is sufficient to support W ARS 1 specific loading of Orc2p and Mcm2p, whereas Cdc6p
requires 3mM ATP for specific loading. Because Mcm2p was loaded onto W ARSl in
the presence of 300[iM ATP, some Cdc6p in these reactions was competent to load
Mcm2p. This may have been a small proportion of the total Cdc6 protein, the rest being
in an inactive conformation that bound non-specifically to the DNA/Dynabeads. In
comparison, the association of Cdc6p to W ARSl specifically, observed at 3mM ATP,
may indicate more complete activation o f the population of Cdc6p at this ATP
concentration.
In summary the addition of the ATP regeneration system had a significant effect
on the efficiency of loading of the components of the pre-RC. In the presence of the ATP
regeneration system, 300p.M ATP or less, was sufficient for pre-RC loading at detectable
levels. However, without the ATP regeneration system, between 3mM and lOmM ATP
was required for loading of Mcm2p. This suggests that without the ATP regeneration
system, the concentration of ATP is reduced during the incubations. The ATP
regeneration system may have affected the ATP concentration in two way. Firstly,
because cell extracts contain many different proteins, even after ammonium sulphate
precipitation, many factors independent of pre-RC assembly will bind to ATP and
hydrolyse, or otherwise reduce its concentration. The Creatine phophokinase catalyzes
the production of ATP, so may have served as a balance to these reactions. Alternatively
the Creatine Phosphate in the regeneration system may have inhibited phosphatases and
thus, prevented some of the consumption of ATP. This experiment has shown that the
ATP regeneration system is essential to maintain the concentration of ATP and, thus, to