The nuclear extract contained > 85% of the total nuclear PMLA and > 75% of the total nuclear DNA polymerase activity in the standard assay.. To measure the inhibitory activity of nuclear
Trang 1The DNA-polymerase inhibiting activity of poly(b- L -malic acid)
Sabine Doerhoefer1, Christina Windisch1, Bernhard Angerer1, Olga I Lavrik2, Bong-Seop Lee1
and Eggehard Holler1
1
Institut fu¨r Biophysik und physikalische Biochemie, Universita¨t, Regensburg, Germany;2Novosibirsk Institute of Biorganic Chemistry, Siberian Division of the Russian Academy of Sciences, Novosibirsk, Russia
The naturally synchronous plasmodia of myxomycetes
synthesize poly(b-L-malic acid), which carries out
cell-spe-cific functions In Physarum polycephalum, poly(b-L-malate)
[the salt form of poly(b-L-malic acid)] is highly concentrated
in the nuclei, repressing DNA synthetic activity of DNA
polymerases by the formation of reversible complexes To
test whether this inhibitory activity is cell-cycle-dependent,
purified DNA polymerase a of P polycephalum was added
to the nuclear extract and the activity was measured by the
incorporation of [3H]thymidine 5¢-monophosphate into acid
precipitable nick-activated salmon testis DNA Maximum
DNA synthesis by the reporter was measured in S-phase,
equivalent to a minimum of inhibitory activity To test for
the activity of endogenous DNA polymerases, DNA
syn-thesis was followed by the highly sensitive photoaffinity
labeling technique Labeling was observed in S-phase in
agreement with the minimum of the inhibitory activity The
activity was constant throughout the cell cycle when the
inhibition was neutralized by the addition of spermidine
hydrochloride Also, the concentration of poly(b-L-malate) did not vary with the phase of the cell cycle [Schmidt, A., Windisch, C & Holler, E (1996) Nuclear accumulation and homeostasis of the unusual polymer poly(b-L-malate) in plasmodia of Physarum polycephalum Eur J Cell Biol 70, 373–380] To explain the variation in the cell cycle, a periodic competition for poly(b-L-malate) between DNA polym-erases and most likely certain histones was assumed These effectors are synthesized in S-phase By competi-tion they displace DNA polymerase from the complex of poly(b-L-malate) The free polymerases, which are no longer inhibited, engage in DNA synthesis It is speculated that poly(b-L-malate) is active in maintaining mitotic synchrony
of plasmodia by playing the mediator between the periodic synthesis of certain proteins and the catalytic competence of DNA polymerases
Keywords: poly(malic acid); cell cycle; S-phase; DNA syn-thesis; histones
Poly(b-L-malic acid) consists ofL-malic acid units, which are
covalently linked by ester bonds between the hydroxyl
group and the carboxyl group in the b position, while the
carboxyl group in a position points away from the polyester
chain [1] The ionized form of the polymer, poly(b-L-malate)
(PMLA), amounts to high concentrations comparable to
DNA in the naturally synchronous nuclei of the
plasmo-dium, the giant polynuclear cell form of the slime mould
Physarum polycephalum[1–3] This organism differentiates
into several cell forms during its life cycle (e.g spores and
amoebae) [4], but only the plasmodium produces
poly(b-L-malic acid) In contrast to the giant cell dimensions, the
billions of nuclei display cyclic events, such as mitosis and
DNA replication, with a high degree of natural synchrony Because of these features, the plasmodium is suited for studying molecular biology of the cell cycle One particular question is the organization of the catalytic competence of DNA polymerases in the context of synchrony
Poly(b-L-malate) was discovered by its activity to bind and reversibly inactivate the endogeneous DNA polymerase
a [1,5] The other replicatively active DNA polymerases (types d and e) have also been shown to bind and become inactivated, whereas the putative repair enzyme, DNA polymerase b-like, was not inhibited [6,7] Binding experi-ments with synthetic polyanions, which differed from PMLA in the distance between the negative charges, demonstrated that specificity of binding is attributed to the particular distance between the negative charges in PMLA [5] This distance is similar to that between phosphate groups in the nucleic acid backbone, in agree-ment with the competitive binding of PMLA and DNA to the polymerases The molecular mimicry suggested that PMLA could bind to histones and to other DNA interact-ing proteins Indeed, large complexes of PMLA not only with DNA polymerases but also with histones and other proteins have been found under conditions close to in vivo [2,7] The binding to histones has been further investigated
by in vitro experiments [5]
If histones and DNA polymerases are together, they are prone to compete for the binding to PMLA The periodic
Correspondence to E Holler, Institut fu¨r Biophysik und physikalische
Biochemie der Universita¨t Regensburg, D-93040 Regensburg,
Germany Fax: + 49 941943 2813, Tel.: + 49 941943 3030,
E-mail: eggehard.holler@biologie.uni-regensburg.de
Abbreviations: AFBdCTP,
exo-N-{[[((4-azido-2,3,5,6-tetra-
fluorobenzylidene)hydrazino)carbonyl]butyl]carbonyl}deoxycytidine-5¢-triphosphate; PMLA, poly(b- L -malate).
Enzymes: DNA polymerase (E.C 2.7.7.7); benzonase (E.C 3.1.21.1).
Note: a website is available at http://www.biologie.uni-regensburg.de/
Biophysik
(Received 8 October 2001, revised 27 December 2001, accepted
7 January 2002)
Trang 2production of histones (or of any other PMLA-binding
molecule) in S-phase could evoke a cycling of free DNA
polymerases and DNA synthetic activity, although the
individual levels of PMLA and DNA polymerases need not
vary For a proper understanding of the role of PMLA it
was thus interesting to know its inhibitory activity over the
cell cycle Because the inhibitory activity could not be tested
under in vivo conditions, experiments were carried out with
nuclear extracts The results were consistent with the
assumption that PMLA was a mediator between increased
concentrations of certain nuclear constituents and the
competence of DNA polymerases in DNA synthesis during
S-phase
M A T E R I A L S A N D M E T H O D S
Materials
Microplasmodia of P polycephalum, strain M3CVIII
(ATCC 96951), were grown in shaken cultures at 27°C, as
described previously [8] Macroplasmodia were obtained as
surface cultures on filter paper by the fusion of
micro-plasmodia, as described previously [9] The mitotic stages
were identified by phase-contrast microscopy [10] One gram
of wet plasmodia corresponded to 2 · 108nuclei DNA
polymerase a (110 UÆmL)1) was purified from plasmodia as
described previously [11] DNase-I-activated salmon testis
DNA for the standard DNA polymerase assay and for
photoaffinity labeling was prepared as described previously
[12] Rabbit antiserum against DNA polymerases type a
and type e, was prepared with a mixture of the purified
P polycephalum DNA polymerases [13], and rabbit
anti-serum against DNA polymerase d by immunization with
synthetic peptides of the enzyme [6] Peroxidase-coupled
anti-(rabbit IgG) Ig was purchased from Pierce Proteinase
inhibitors were used in a cocktail of the following
concentrations after dilution with the extracts: 5 mMsodium
bisulfite, 0.2 mM phenylmethanesulfonyl fluoride, 1 mM
benzamidine (Sigma), 1 lMpepstatin A (Merck), 10 lM
leu-peptin (Sigma), 1 mgÆmL)1 aprotinin (Merck), 10 lM
tosyl-L-lysine chloromethyl ketone (Calbiochem), 100 lM
pefablock SC (Merck), and 2 lgÆmL)1 E 64 (Boehringer
Mannheim) For photocrosslinking, the dCTP analogue
exo-N-{[[((4-azido-2,3,5,6-tetrafluorobenzylidene)hydrazi-no)carbonyl]butyl]carbonyl}deoxycytidine-5¢-triphosphate
(AFBdCTP) was prepared as described previously [14], and
was a gift from Safronov (Novosibirsk) Benzonase grade II
(25 000 UÆmL)1) was purchased from Merck
Nonradio-active dNTPs and standard proteins for SDS/PAGE
were obtained from Pharmacia (Sweden) [3H]dTTP
(60 CiÆmmol)1, 1 Ci ¼ 37 GBq) and [a-32P]dATP
(3000 CiÆmmol)1) were purchased from Amersham
Preparation of nuclear extract
Nuclei were prepared either from macroplasmodia
follow-ing their third mitosis, or from microplasmodia harvested
after 2 days of inoculation The plasmodia were washed by
centrifugation (500 g, 10 min, 4°C) in cold water,
suspen-ded in disruption buffer (2 g of solvent per 1 g of wet
plasmodia; 15 mMTris/HCl pH 7.5, 5 mMEGTA, 0.5 mM
CaCl2, 15 mMMgCl2, 500 mMhexylenglycol, 10% dextran,
14 mM2-mercaptoethanol, and protease inhibitor cocktail)
and disrupted in a Dounce homogenizer (10–12 strokes) Nuclei were pelleted over a 25% Percoll gradient in the above buffer, as described previously [2] The pellet contained 2 ± 1· 108nuclei per g of wet microplasmodia Nuclear extracts were prepared by incubating for 10 min on ice in an equal volume of extraction buffer (final concen-trations 50 mM Tris/HCl pH 7.5, 0.3M KCl, 20 mM
MgCl2, 0.5% Triton X-100, 20% glycerol, 1 mM 2-merca-ptoethanol, protease inhibitor cocktail) and centrifugation
at 700 g The nuclear extract contained > 85% of the total nuclear PMLA and > 75% of the total nuclear DNA polymerase activity in the standard assay Results by SDS/ PAGE and Western blotting with specific antisera against DNA polymerases a and e [13], and DNA polymerase d [14] were consistent with the recovery of > 95% of DNA polymerase a and > 75% of the other DNA polymerases in the extract
Standard DNA polymerase activity and inhibition assays Total DNA polymerase activity was assayed as described previously [12] The standard assay contained in 150 lL
50 mM 3-(N-morpholino)propanesulfonic acid/potassium salt (pH 7.5), 50 mMKCl, 10 mMMgCl2, 3 mM EDTA,
3 mM 2-mercaptoethanol, 33 lM each of dATP, dCTP, dGTP, 3 lM [3H]dTTP (1 CiÆmmol)1), 20 lg DNase-I activated salmon testis DNA, 80 lg bovine serum albumin, and DNA polymerase After a 30-min lcubation at 37°C, 10% (v/v) saturated cold trichloroacetic acid in water was added and the precipitate collected on Whatman GF/C filters, which were washed with trichloroacetic acid, then with 70% (v/v) ethanol/H2O dried, and counted with 20% efficiency If required, either 0.4 mM spermineÆ4HCl or
2 mM spermidineÆ3HCl were included to suppress binding and inhibition of polymerases by PMLA [1] One unit of polymerase activity is equivalent to the amount of enzyme that catalyses the incorporation of 1 nmol nucleotides during 1 h
The same conditions were used in the inhibition experi-ments, except that the biogenic amines were omitted To measure the inhibitory activity of nuclear extracts during the cell cycle, 0.4 U of purified DNA polymerase a was present
in the assay under the above standard conditions comparing the reaction rates in the presence (vi) and the absence (reference, vo) of nuclear extract equivalent to 1.5· 105
nuclei To account for effects of particular ingredients in the extract buffer, appropriate amounts of these reagents were added to the reference reaction mixture The low poly-merase activity contained in the extract due to DNA polymerase b-like (which was not inhibited by PMLA) was measured in parallel and subtracted from the crude value for vi The inhibitory activity is defined in terms of the reciprocal value of the inhibition constant Kiÿ 1 ¼ [E–PMLA]/[E]Æ[PMLA] The concentration of the poly-merase–inhibitor complex, [E–PMLA], can be expressed in terms of [E]o) [E] The concentration of free polymerase, [E], and of the total polymerase [E]ois proportional to viand
vo The expression for the relative inhibitory activity is then (vo) vi)/vi ¼ [PMLA]/Ki Therefore, the higher the con-centration of free PMLA, and the lower the value of the inhibition constant (Ki), the higher the relative inhibitory activity (vo) vi)/vi In the presence of ligands that bind com-petitively to PMLA, the value of K increases as a function
Trang 3of rising concentrations and affinities of these ligands For
example, if spermine hydrochloride, which binds to PMLA,
is present, the inhibition may be totally neutralized The
reciprocal of the direct values of the relative inhibitory
activity will be shown in the results, as they correlate directly
with the degree of residual DNA polymerase activity
DNA replication of single macroplasmodia was followed
under in vivo conditions at various phases in the cell cycle
The plasmodia were grown on filter paper for a particular
period of time in the cell cycle One fourth of the
plasmodium, usually 80 lg, was transferred to fresh
medium containing 5 lCiÆmL)1 [methyl-3H]thymidine
(25 CiÆmmol)1) and grown for 15 min at 27°C The
remainder was allowed to grow and used as the source of
further samples The incubation was terminated by fixation
in a 10-mL solution containing ice-cold 5% saturated
trichoroacetic acid and 50% acetone The samples were
disrupted in a Dounce homogenizer and filtered on GF/C
After washing with trichloroacetic acid/acetone and
etha-nol, the filters were dried, and the radioactivity was counted
in a scintillation cocktail
Photoaffinity labeling of DNA polymerases
Labeling of DNA polymerases was carried out in a 20-lL
solution containing 5 lL nuclear extract, 3–5 lM[32P]dATP
(3000 CiÆmmol)1), 125 lM AFBdCTP, 33 lM of each
dGTP and dTTP, 50 mM3-(N-morpholino)propanesulfonic
acid buffer (pH 7.5), 50 mM KCl, 10 mM MgCl2, 3 mM
EDTA and 5 lg activated salmon testis DNA [7]
Escheri-chia coliDNA polymerase I served as a positive control in a
parallel, but otherwise identical, reaction mixture In other
control reactions, to exclude staining due to DNA
poly-merase adenylation or phosphorylation, the photoreactive
nucleotide was omitted The polymerization reaction was
carried out in the dark for 10 min at 37°C An aliquot was
then irradiated for 2 min Free DNA and DNA protruding
from crosslinked complexes with proteins were digested
with benzonase (25 U per sample) for 10–15 min at 37°C
The sample was heated for 3 min with Laemmli buffer [15]
and examined by denaturating SDS/PAGE (10%
poly-acrylamide gel) Proteins were electroblotted onto Millipore
Immobilon membranes [16] and visualized by
autoradio-graphy (Kodak X-OMAT LS) at )70° (5–7 days) The
identities of blotted proteins were verified by
immunostain-ing of the same membranes Intensities of bands were
quantified with a Boehringer Mannheim Lumi ImagerTM
The intensity of labeled E coli DNA polymerase I in the
same gel served as a reference
R E S U L T S
Finding optimal assay conditions to measure
the inhibitory activity in nuclear extracts
In previous analytical and preparative experiments, it has
been shown that PMLA was the constituent that
specific-ally inhibited replicative DNA polymerases in nuclear
extracts [1,2,7] In the present study, we measured the
inhibitory activity of PMLA using purified DNA
poly-merase a of P polycephalum as an added reporter To find
the optimal assay conditions, the extracts were prepared
from the nuclei of microplasmodia that naturally included
all phases of the cell cycle As considered in Materials and methods, the degree of inhibition depends on both the concentration of free PMLA and the inhibition constant This parameter reflected the affinity of the polymerase and both the affinity and concentration of competing ligands for binding to the polyanion In the (added) nuclear extract, such ligands were histones, and probably other DNA-binding proteins [2] To obtain an optimal response by the reporter polymerase to a varying inhibitory activity in the nuclear extract, the amounts of the added polymerase and extract had to be optimized To this end, the titration of a fixed amount of nuclear extract, corresponding to 1.5· 105 nuclei, was performed in the first experiment (Fig 1) In the beginning of the titration, the polymerase activity remained suppressed until the inhibitory activity was neutralized by
an amount of 0.38 ± 0.03 U of the reporter DNA polymerase (the arrow in Fig 1) During continued addi-tion of the polymerase, the enzyme activity increased in parallel with the activity of the control experiment in the absence of extract An amount of 0.4 U of the reporter DNA polymerase, close to the neutralization point, was chosen for the measurement of the inhibitory activity during the cell cycle
We have previously shown that the inhibitory activity of purified PMLA is neutralized in the presence 0.4 mM
spermine hydrochloride [1] To confirm that PMLA was the only inhibitor of DNA polymerases in the extract above,
we measured the polymerase activity in the presence of added spermine hydrochloride A value of 1.2 ± 0.03 U (five measurements) was observed and referred to the endogenous DNA polymerases The experiment was repea-ted with the extract containing in addition 0.4 U reporter DNA polymerase a An amount of 1.6 ± 0.03 U was measured in this case (five measurements) The difference of 0.4 ± 0.04 U was in agreement with the added 0.4 U The same results were obtained when nuclear extracts in the S-phase and in G2-phase were compared The agreement was consistent with the assumption that the inhibitory activity was a property of PMLA in the extract
Fig 1 The inhibition of purified DNA polymerase a by poly(b- L -malate) in the nuclear extract (d) The activity was measured as a function of added amounts of DNA polymerase a in the standard DNA polymerase assay that contained the extract of 2 · 10 6 nuclei isolated from microplasmodia (j) The control in the absence of the nuclear extract containing an equivalent of the ingredients in the nuclear extraction buffer The arrow refers to the equivalence of the added DNA polymerase activity and the inhibitory activity.
Trang 4The inhibitory activity during the cell cycle
The inhibitory activity during the cell cycle was measured in
the presence of 0.4 U of (added) purified DNA polymerase
a and the extract of 1.5· 105 nuclei The dependence is
shown in Fig 2A in terms of the reciprocal values,
corresponding to the residual DNA polymerase activity
The maximum at 1 h after mitosis corresponded to a
minimum in inhibitory activity and a maximum in the
residual polymerase activity (63% of the reference activity)
After 2 h following mitosis and during the remainder of the
cell cycle, the polymerase activity approached a basal level
of 10–20% of the reference activity
The activity of DNA polymerases measured
by photo affinity labeling
According to Fig 2A, the inhibitory activity in the nuclear
extract showed a minimum between 0 and 2 h after mitosis
(Fig 2A) It was of interest whether this interval coincided with some endogenous residual activity of the DNA polymerases in the nuclear extract (DNA polymerase a not added) Because the (residual) activity of the endo-genous DNA polymerases was too low to be detected with the standard assay, we introduced a highly sensitive technique of affinity photo crosslinking [7] Briefly, the enzymatically active DNA polymerase catalysed the primer elongation with radioactively labeled nucleotides of high specific radioactivity Then, the elongated primers were photo crosslinked to the active polymerases within the elongation complex The amount of radioactivity covalently attached to DNA polymerases was an indicator of the polymerase activity and was measured after SDS/PAGE by autoradiography Separate results are shown in Fig 2C for DNA polymerase e and for DNA polymerases of type a, type-b-like, and type d in Fig 2B, which were not resolved from each other DNA polymerase e showed the highest activity during the first hour after mitosis Then the activity
Fig 2 DNA polymerase activitiy during the cell cycle of macroplasmodia All graphs are drawn to the same scale to facilitate comparison In this scale, the measured value at 0.7 h in the nuclear division cycle is arbitrarily set equal to one unit in each panel M denotes mitosis (A) The reciprocal inhibitory activity v i (v o ) v i ))1(see text) calculated from values of the residual polymerase activity (v i ) and the reference activity (v o ) of added 0.4 U
of purified DNA polymerase a in the standard DNA polymerase assay with (v i ) and without (v o ) nuclear extract The extract of 2 · 10 6
nuclei was prepared from macroplasmodia at various times during the cell cycle Bars refer to standard deviations (three determinations) One unit on scale refers to 1.64 U of the reciprocal inhibitory activity (B) Activity of endogenous DNA polymerases a, d, and b-like in the nuclear extract, measured
in arbitrary units (staining intensity) by the highly sensitive technique of photoaffinity labeling Single types of DNA polymerases could not be resolved Bars refer to standard deviations (three determinations) (C) Activity of endogenous DNA polymerase e, measured in parallel with the DNA polymerases in panel B One unit on scale compares to half a unit in (B) (D) DNA synthesizing activity of plasmodia at various times in the cell cycle The incorporation of radioactivity into acid precipitable material has been measured during a brief exposure to [methyl-3H]thymidine One unit on scale refers to 6 Bq [ 3 H]TMP incorporated (E) The activity of endogenous DNA polymerases in the extract of 2 · 10 8 nuclei at various times during the cell cycle One unit on scale refers to 90 U of DNA polymerase activity (see Materials and methods) The activity was measured in the presence of added 2 m M spermidineÆ3HCl (?) to neutralize the inhibitory activity of PMLA In the absence of spermidineÆ3HCl, activities of DNA polymerase, except of DNA polymerase b-like, are inhibited by PMLA contained in the extracts.
Trang 5decreased and fell to a basal level 2 h after mitosis The
dependence was similar for the unresolved DNA
poly-merases The high basal level was explained by the
contribution of DNA polymerase b-like, which was not
inhibited by PMLA The results show that the minimum in
the inhibitory activity corresponded with a maximum of
DNA polymerase activity in the cell cycle
Thein vivo activity of DNA polymerases
The cell cycle dependence of DNA polymerase activity was
followed under in vivo conditions The incorporation of
radioactivity into DNA was determined following a brief
exposure to [methyl-3H]thymidine The results in Fig 2D
show a maximum in activity of DNA synthesis at 1 h after
mitosis, followed by a decrease approaching a basal level at
2 h after mitosis Thus, the in vivo and in vitro activities of
DNA synthesis corresponded with the minimum of the
inhibitory activity in the cell cycle
The activity of DNA polymerases in the nuclear extracts
after neutralization of the inhibitory activity
by spermidine hydrochloride
Although the appearance of an activity peak of DNA
polymerases was consistent with the minimum in the
inhibitory activity of PMLA, an additional periodic
variation in the intrinsic activities of the endogenous
DNA polymerases was not excluded It has been shown
that biogenic polyamines bind to PMLA and neutralize its
inhibitory activity against DNA polymerases [1] This
finding allowed us to examine whether the intrinsic DNA
polymerase activity of the extract varied during the cell cycle
or whether it was totally modulated by the degree of
complex formation of DNA polymerases with PMLA The
circles in Fig 2E show the DNA polymerase activity of
nuclear extract in the standard assay in the case when
spermidine hydrochloride was not present This activity
referred to DNA polymerase b-like, which was not inhibited
by PMLA [1] and thus did not show a cell cycle dependence
The other DNA polymerases displayed no measurable
activity in the standard assay due to the strong inhibition by
PMLA (see also above) The squares in Fig 2E refer to the
addition of spermidine hydrochloride The activities of the
DNA polymerases were derepressed, because the inhibitory
activity was neutralized The data show the superpositions
for DNA polymerases a–e Importantly, a cell cycle
dependence was not indicated The variations observed in
Fig 2A–C were explained by the change in the inhibitory
activity of PMLA during the cell cycle
D I S C U S S I O N
Myxomycetes comprise a large family of organisms that
typically generate a plasmodium, a polynucleated giant cell
among other cell forms in the life cycle [4] Interestingly, the
billions of nuclei in these syncytia participate with high
degree of synchrony in the division cycle All of the
myxomycetes species so far examined contained PMLA in
the plamodia The PMLA level in the nuclei is high and
comparable to that of chromosomal DNA [1,3] It remains
constant during the cell cycle, and PMLA synthesized in
excess amounts is secreted into the culture medium In
contrast to varying degrees of PMLA synthesis, the content
in the nuclei is conserved among different species (Karl, M., Anderson, R W & Holler, E., unpublished data) The various observations suggest that PMLA may play a role in many biological functions One function has been attributed
to the induction of sporulation of P polycephalum [17] and another to the carriage and storage of DNA polymerases, histones, and other nuclear proteins in the plasmodium [2,18]
In connection with the role as a carrier and storage function, the inhibitory activity of PMLA towards the replicative DNA polymerases was of interest [1,6,7] The coupling of the carrier/storage function and the inhibition of DNA synthesis suggested an effect on the availability of DNA polymerase activity during the cell cycle Our results showed an inverse relation of the inhibitory activity with the activity of the endogenous DNA polymerases in the extract
as well as with the DNA synthetic activity (S-phase) in living plasmodia While the DNA synthetic activity in the nuclear extract was periodic with a maximum in S-phase, the activities of the DNA polymerases in the standard assay were constant after neutralization of PMLA The cell cycle-independent biosynthesis and activity of DNA polymerase a has been described by Western blotting and activity gel analysis [19]
The findings extend the storage/carrier role of PMLA to a controller function of the catalytic competence of DNA polymerases The results in Fig 2E revealed that the activities of DNA polymerases on their own did not vary and that they were inhibited by complex formation with PMLA The inhibition would be permanent unless the complexes dissociated in S-phase The dissociation could be principally controlled by a periodical decrease in the level of PMLA However, the nuclear content of PMLA has been shown to be constant over the cell cycle [3] As a carrier, PMLA binds also histones and other nuclear proteins [2] Core histones, for example, are heavily synthesized in S-phase [20], and are likely to compete with DNA poly-merases for the binding of PMLA Once free, DNA polymerase is competent for DNA synthesis However, the identity of these effectors is still unclear We favor histones, because they are depleted from the PMLA complexes by forming nucleosomes, when their synthesis ceases at the end of S-phase This would explain the end of the activity period of DNA polymerases Instead of assuming a neutralization of the inhibitory activity, the DNA polymerases could bind effectors, which induce the release of polymalate from the complex While this mech-anism is principally possible, it is not supported by experimental evidence PMLA binds to DNA polymerases competitively with DNA [1], and such factors would also inhibit the binding of template-primer DNA and thus the polymerase activity Although the inhibitory activity of PMLA and its cycling has been established in the nuclear extract, it is speculated that a similar mechanism exists in the nuclei of plasmodia A major reason for this assumption is the finding that PMLA forms complexes with DNA polymerases, histones and other nuclear proteins under conditions close to in vivo [2,7]
The PMLA-dependent cycling of DNA polymerases does not account for the timing of DNA replication It also does not take into account the periodic synthesis of factors such
as the proliferating-cell nuclear antigen (PCNA) and
Trang 6replication factor C (RF-C) [7], but merely links the catalytic
competence of DNA synthesizing polypeptides to the
S-phase These factors are recognized only with specialized
template-primers but not with activated salmon testis DNA,
as used here Because PMLA is only found in the
multinucleated plasmodia and not in the mononucleated
amoebae, it is speculated that the periodic change of the
inhibitory activity is involved in the maintenance of the
plasmodial synchrony by coordinating the catalytic
competence of DNA polymerases throughout the giant
cell We are currently investigating the transfer of PMLA
between the nuclei, and the competitive exchange of DNA
polymerases and effector proteins
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