CONTRIBUTIONS TO DNA and RNA POLYMERASE KINETICS
JACQUES NINIO
( included in
the web site
http://www.lps.ens.fr/~ninio
)
TOPICS DISCUSSED
HERE:
NTP cleavage
reactions.
Polymerization/excision kinetics.
Mnemonic effects.
Exonuclease
warm-up.
SEE IN OTHER WEB
CHAPTERS
More on the next
nucleotide
effect (Contributions to the kinetic theory of accuracy).
The
peelback effect
(Contributions to mathematical biology).
Mutation strategies (in
molecular
evolution section).
Non-enzymatic replication (Contributions to the
origins
of life).
++++++++++++++++++++++++++++++++++++++++++++++++++++++++
The existence of a
proofreading activity associated with E. coli or phage T4 DNA
polymerase was
revealed in 1972 [1, 2]. I was not immediately aware of this work, and
was
mostly concerned with ribosomal accuracy, trying to interpret Gorinis
observations on high and low accuracy ribosomes in terms of changes in
kinetic
parameters of the amino acid incorporation reaction. When this work was
sufficiently advanced, and I had worked out the (rather elementary)
mathematics
needed to understand how changes in the kinetic parameters of an enzyme
reaction impinged on its accuracy [3], I turned to DNA polymerases. In
the
beginning, I spent much time on two articles from Kornbergs
laboratory. The
first [4] gave the affinities of Kornbergs polymerase for the four
deoxynucleotide triphosphates in the absence of template. The natural
preferences were ranked as follows :
G > A > T > C . I was thinking of using these results
in
relation to base pair energetics (the DNA polymerase was perhaps using
these
differential binding affinities for counteracting some potential
errors) or in
relation to the nucleotide substitution tables derived from
phylogenetic
studies. I failed to derive any useful insight from this work. The
second was a
very astute, historically important article, in which Josse, Kaiser and
Kornberg [5] made an analysis of the nearest neighbours, in the DNA
strand
being replicated, of each of the four incorporated nucleotides. I hoped
to use
these nearest-neighbour data to have a better understanding of
replication
energetics, but here also, I failed to derive any useful insight. When
the
first papers on the proofreading function appeared, I remember that
there was
some disagreement between Hershfield [6] who was claiming that the DNA
polymerase fidelity mutants were essentially altered at the
incorporation level
and Bessman [2] who spoke of the ratio between the incorporation and
the
excision activties of the DNA polymerase. From my own experience with
ribosome
kinetics [3] I knew that accuracy was governed by ratio of rates, and
that
distinguishing between forward and backward effects was not an easy
task, so I
did not have a preference for incorporation or excision effects.
Another
article fell into my hands, in this period (I was in Orgels laboratory
at the
Salk Institute), and this one did inspire me. In this article, Hall and
Lehman
described puzzling observations [7]. The authors were studying T/G
misincorporation errors made by a standard and a mutagenic DNA
polymerase from
phage T4 in the in vitro
replication of Poly(dC). Surprisingly, they found that they could not
dilute
the T misincorporations by increasing the concentration of dGTP at
constant
TTP. Instead, the T/G ratio seemed to reach an asymptotic value. This
odd
result was not even mentioned in the rather inocuous summary of their
article.
The article had been published in 1968, at a time in which the
proofreading
function was still unknown. Reading this article in 1973, I almost
immediately
saw that Hall and Lehmans results had to do with the balance between
nucleotide incorporation and excision at various nucleotide
concentrations. It
was childplay to transpose my ribosome kinetic analysis [3] to the
incorporation/excision balance.
I
thus derived the next nucleotide effect according to
which
incorporation errors at any position would increase with the
concentration of
the nucleotide to be incorporated next. I did not hurry to publish it
because
on one hand it seemed to be a modest step forward with respect to [3],
and on
the other hand I had the hope that my colleagues in Chapevilles
laboratory in Paris
would perform the experiments, and prove the phenomenon. I also found
in a work
by Howard and Tessman [8] indirect evidence for the next nucleotide
effect in
vivo. (With modern
technologies, the
Howard and Tessman kind of experiment may produce superb results). I
wrote a
note, in French, entitled commentaires sur une exprience de
Hall et
Lehman in which I made a mathematical analysis of Hall and
Lehmans
data, and sent the note to Gilbert Brun, in Chapevilles laboratory.
The note
also contained kinetic calculations on what I expected when
misincorpotation
was due to a tautomery. (In this case, proofreading becomes useful only
after
the return to the standard conformation of the nucleotides facing each
other,
giving rise to a quadratic effect in error correction). However, either
my
colleagues did not understand what was at stake, or they were not
interested
enough in the subject of DNA polymerase accuracy, so nothing came out,
experimentally, from their hands.
Ultimately,
I mentioned the next-nucleotide effect in various places. For instance,
it is
mentioned as follows, in [9], page 593 :
Discrimination
being related to the time which separates two incorporations, it should
depend
on the concentration of the dNTPs. More precisely, the errors of
incorporation
at any position along the chain would increase with increasing concentrations of that dNTP which is to
be
incorporated at the next
position. Such an effect can in principle be revealed in classically
designed
experiments [29, 30],
References
29 and 30, in the above paragraph were to Hall and Lehmans and to
Howard and
Tessmans papers [7, 8]. I did not say more, because this effect was
quite
obvious, within the context of the more sophisticated preceding
developments in
the same article. On this topic, see also the website chapter on Contributions to the kinetic
theory of accuracy .
At
some point during the Sept 72 to March 74
period at the Salk Institute, Leslie Orgel was contacted by
David Galas,
who was then working with Elbert Branscomb on translation error
propagation in
E. coli. I do not remember whether or not he visited the lab in person,
but
this was the beginning of a correspondence between David and I, on
theoretical
treatments of error-feedback, and later on DNA polymerase kinetics (see
the
website chapter contributions to the kinetic theory of
accuracy ).
My
article on ribosome kinetics [3] was submitted in July 1973 and after
that, I
must have started trying to go one step further and construct kinetic
amplification mechanisms. After the sparkle, the very last days of
December
1973, I must have tried to extend the concept of kinetic amplification
to the
workings of a classical proofreading function. As I mentioned above, I
had
already in mind the notion that it was useful to wait, in the case of
tautomeries, before exerting the proofreading activity. I soon found
that in
the standard case, the shape of the time-window allocated to
proofreading was
extremeyl important, and that what really mattered was how it ended.
More
precisely, I found that the substrate (the base-pair being proofread)
must not
be allowed to escape from the proofreading function in a single
elementary
step, but must be held under attack for at least two steps. This was my
delayed-escape mechanism, which has not yet received much
attention, although I think that there is much evidence in its favour
in the
case of aminoacyl-tRNA ligases. The experiments made in my group on
polymerization/excision kinetics were for some homopolymers consistent
with a delayed
escape mechanism, and for others they did not show the kinetic
complexity
expected from these mechanisms (see the section below on
polymerization/excision kinetics)
Upon
my return in France, in April 74, I tried to initiate work on catalysis
by randomly
generated peptides (see the website chapter contributions
to the
origins of life ). At the end of 1974, I read John Hopfields
article on
kinetic proofreading [10] and saw immediately that when applied to DNA
or RNA
polymerases, his scheme made predictions which were radically different
from my
expectations, based on either the next nucleotide effect, or the
delayed escape
mechanisms. I must stress again the point that although there is a
striking
convergence between Hopfields proposals [10] and mine, there are also
differences, and these become rather important, when one tries to
confront the
predictions with experimental reality. (See for instance my recent
analysis of
the GTPase reactions on the ribosome [11]). These differences have been
obscured,
in part because John Hopfield chose to make his arguments general, and
incorporated classical proofreading in his conception of kinetic
proofreading.
On my side, I chose to make a sharp distinction between classical and
kinetic
proofreading. Classical proofreading is what we learnt from the studies
of
Kornberg, Bessman and others, e.g., [1, 2, 6]. After making the
internucleotide
bond, a DNA polymerase may excise the last nucleotide it had
incorporated. Now,
there was a new idea in Hopfields article which was not in my article,
namely
that in the incorporation pathway, the dNTP could be cut into dNMP and
pyrophosphate, and the dNMP could be rejected BEFORE having a chance to
be
linked to the preceding nucleotide. Then, dNMP would be produced in
proportion
to the corresponding dNTP, and would not vary with the concentration of
the
next nucleotide.
This
time, I convinced my colleagues in Franois Chapevilles laboratory to
look
seriously at the polymerization and excision kinetics. Kinetic
experiments were
performed. Thin layer chromatography was used to separate the various
products
of the reactions. It turned out that nucleoside diphosphates were
produced, in
direct proportion to the nucleoside triphosphates. So, I thought, we
could have
here a variant of Hopfields scheme, in which the incoming dNTP would
be split,
prior to incorporation, into dNDP and phosphate [12]. The work was
received
with skepticism. I now believe that these cleavage reactions are
biologically
relevant companions of the polymerase activities, and that they have a
role in
accuracy, but not the one I had imagined (See section below on the NTP
or dNTP
cleavage reactions).
After
the NDP and dNDP episode,
Franoise Bernardi and I returned to the quantification of the
dNMP
produced during polymerization reactions, and publlished in fact the
first
kinetic experiments showing how the polymerization/excision ratio
varied with
the concentration of the correct nucleotide [13]. (See further down the
section
on polymerization/excision kinetics). The work was pursued by Mohammed
Saghi
and Mireille Dorizzi, then took a new direction with the arrival of
Catherine
Papanicolaou. We had found strange variations in the kinetics with the
concentrations of the reactants, and also with the concentration of the
catalyst, the DNA polymerase itself. This led to a series of articles,
initiated in [14] in which we demonstrated that E. coli DNA polymerase
I had a
mnemonic behaviour. This was both interesting and frustrating, because
the
mnemonic behaviour made it very difficult to establish the mechanism of
the
reaction at the level of detail which would have satisfied me. (See
section
below on mnemonic effects and exonuclease warm-up).
After
her thesis, Catherine Papanicolaou went to the USA and worked with Lynn
Ripley.
Philippe Lecomte, who had collaborated with her on the mnemonic effects
was
progressively discouraged, and our last experimental paper on the topic
was
published in 1988. Nevertheless, Tom Kunkel and Roel Schaaper were kind
enough
to invite me to a meeting they were organizing in North Carolina in
September
1989, on The fidelity of DNA synthesis : structural and
mechanistic
perspectives . By then, DNA polymerase crystals had been obtained
and
interpreted in the laboratory of Tom Steitz [15], and fast kinetic
experiments
were being performed in Stephen Benkovics laboratory [16]. There were
two
major findings which I had not anticipated. First, until then, I had
conceptualized the bifunctional polymerases as having a head which
incorporates
nucleotides, and a proofreading function located behind. However, the
crystal
structures demonstrated that the exonuclease activity was located ahead
of the
incorporation function. Accordingly, after the formation of the
internucleotide
link, the growing terminus and the portion of DNA being copied had to
slide
forward to reach the exonuclease site. These mechanistic details did
not
threaten the kinetic treatments leading for instance to the
next-nucleotide
effect, since these treatments were rather general, and were not tied
to
precise mechanistic details. On the other hand, it was also the case
that I
hade never taken into consideration this arrangement with an
exonuclease
activity ahead of the polymerase activity. I still blame myself for
this lack
of lucidity.
There
was an enormous difference of scale, between my meagre group, and the
American
teams. Not only they had budgets which were orders of magnitude above
mine, but
also they had an almost unlimited access to students manpower, which
was
unthinkable under the constraints of the French system. Although I had
some
confidence in my capacity to produce astute work, I felt at this
congress that
we were entering an era in which scientific truths in biology could be
negociated between big laboratories. So, there was no room for
independent
work. After publishing as a swan song my transient
mutator
hypothesis [17], I resigned from my position of group leader in
molecular
biology, and joined a team of physicists interested in cognitive
sciences at
Ecole Normale Suprieure.
+++++++++++++++++++++++
NTP or dNTP
cleavage
reactions
In
1974, several members of Franois Chapevilles laboratory were working
on DNA
polymerases. The most active in the field, among them was Gilbert Brun
who was
mainly working on eukaryotic DNA polymerases. Franois Rougeon was also
there,
and was working on reverse transcription and its biological
implications.
Franoise Bernardi joined the lab and teamed with me in the summer, and
she
started a project on looking for catalytic activities in randomly
synthesized
peptides (see the website chapter of contributions to the
origins of
life ). The publication of
John Hopfields paper (which we received quite late, due to a postal
strike)
incited me to have a close look at DNA polymerase kinetics, and in
particular,
see whether the deoxnucleoside monophosphate was produced, in a
classical way,
through exonucleolytic proofreading, or as envisioned by Hopfield,
through a
splitting of the dNTP into dNMP and pyrophosphate, prior to the
formation of
the internucleotide linkage.
Franoise
Bernardi initiated the experiments, possibly in January 1975 (see the
website
chapter on contributions to the kinetic theory of accuracy). We
followed
the kinetics by spotting each extract from the reaction mix on a
thin-layer
chromatography sheet, and separating the various products of the
reaction. It
turned out that, in addition to the appearance of dNMP, we found that
substantial amounts of dNDP (nucleoside diphosphates) were produced
during the
reaction. I was very excited, because I thought that the dNDP could be
produced
according to a variant of Hopfields scheme, in which the dNTP was
split into
dNDP and phosphate prior to the formation of the internucleotide bond.
The
production of dNDP was observed with E . coli DNA polymerase I
(and, I
think, with one or two eukaryotic DNA polymerases), and the production
of NDP
was observed with E. coli RNA polymerase. The kinetics of appearance of
these
nucleoside diphosphates were in remarkable agreement with the
predictions I had
made on the basis of the modified Hopfield scheme. However Chapeville
was not
convinced by the arguments. He had in mind an enzymologists golden
rule
stating that enzymes which produced posphate from nucleotides were
quite
distinct from those which produced pyrophophate. A same enzyme could
not
produce both, as implied by my reaction scheme. Nevertheless, I was
enthousiastic and convincing, and by March 21st, 1975 we submitted to
Franois
Chambon acting as editor of FEBS Letters, a manuscript describing our
work. The
paper was accepted, after making small modifications. It appeared in
the
September 1975 issue of FEBS Letters [12].
The
least I can say is that it did not convince other workers in the field.
Soon
afterwards, a Russian team described the separation of a NDP generating
activity from the polymerising activity in RNA polymerase. This was a
blow, but
it left the door open for different reaction schemes in which NDP could
have
been a side product. Note that in eukaryotic DNA polymerases, the
polymerising
activity and the exonucleolytic proofreading activities are on
different
subunits of the enzme (e.g., [18]) so the separability of the two
activities
does not imply that they should be functionally unrelated. There was
however another
serious problem with our paper. I did not know, at the time of the
work, about
radioactivity quenching problems. The quantitative arguments which I
had
derived, in the case of RNA polymerase, from comparative measurements
of
tritium and P32 radioactivity did not take into account the
differential quenching effects of the two radioactive labels. Once they
were
taken into account, much of the strength of the argument was removed,
and
alternative, less interesting interpretations could be found. I
acknowledged
this state of affairs, writing for instance in [19] :
In an
earlier work,
we reported that both DNA and RNA polymerases were able to degrade
dNTPs into
dNDPs and phosphate (Ninio et al., 1975). Because of a counting
artefact, the
quantitative relationships given in the paper were not correct, so that
the
relationship between these activities and kinetic amplification is
doubtful.
With the progress in the chemical synthesis of DNAs
of defined sequences, we hope to clarify the point in
the near future.
Remaining
with RNA polymerases, the NDP producing activiy came to the front scene
again
when a low fidelity mutant of RNA polymerase was isolated [20].
Jonathan
Gallant and Randell Libby found that the genetic defect coincided with
a loss
of the activity converting NTP into NDP [21-23]. They assigned this
activity to
the b subunit of
RNA polymerase. Libby came
for a short period in my lab, in Paris, to perform some kinetic
experiments,
taking advantage of the competence of Philippe Lecomte in this domain.
At that
time, I was paying great attention to diffusion reactions, and to the
implications of in vivo
viscosity. Assume that an incorrect nucleotide has been rejected by a
polymerase. Will it diffuse away immediately, or should we expect the
nucleotide
to hang around the binding site, and have several additional
opportunities to
be handled again by the polymerase at the same site ? I thus
thought that
one way to reduce the danger would be to label the
expelled
nucleotide, so that it could not be re-used immediately.
My concern for the implications of
viscosity was expressed in [19] :
The
passage from the highly viscous conditions of the cells interior to
the liquid
conditions of in vitro
experiments may affect quite differently the various kinetic constants
of a
reaction. A polymerase will wait longer for the incoming nucleotide,
but if it
dissociates from the template, it might fail to diffuse away, and might
reassociate readily, the more so as imprinting effects on the template
may last
longer. Side products of the reaction like pyrophosphate or
deoxynucleoside
monophosphate might hang around and be in abnormally high local
concentrations,
with respect to usual in vitro
assays. Is this the reason why RNA polymerase carries a companion
activity
which eliminates pyrophosphate by an unusual method (Volloch, Rits and
Tumerman, 1979) ? .
The
reaction described by Volloch et al.
[24, 25] is the production of trimetaphosphate (PPPi) and
phosphate from
two pyrophosphate (PPi) molecules. There was also a report, by
Hinrichsen et
al. [26] of the synthesis of dinucleoside tetraphosphates by RNA
polymerase B
from calf thymus, according to the reaction pppN + pppN pppNpN + pp.
When
Randell Libby came, I proposed to him to study the effect of viscosity.
I
believed that NDP formation had to do wih a labelling of the
nucleotides which
had been rejected. Unfortunately, there were technical problems with
the
preparations or the assays, which could not be solved within the time
allocated
for Libbys visit, and since Libby left the subject afterwards, I do
not know
what to think about all that. The proposals of Libby and Gallant were
challenged by Butzow et al. [27] who wrote :
The
greater cleavage with noncomplementary DNA occurs because of the lack
of
incorporation by the polymerase, which then does not compete with the
NTPase
for the substrate pool. Thus, these findings indicate that the cleavage
preference of the NTPase for noncomplementary NTPs is not part of a mechanism for error prevention
during
transcription .
The
same interpretation applied to my early work [12], once the
radioactivity
quenching artefact was removed. On the other hand, while such
interpretations
are compatible with in vitro
kinetics, they relegate a number of biological observations to the rank
of mere
oddities : (i) why should there be NTPase activities at all ?
(ii)
why these activities should be so hard to separate from polymerising
activities ? (iii) what is the biochemical basis for the RNA
polymerase accuracy
mutants studied by Libby and Gallant ?
I
consider that there might well be a link between the above mentioned
parasitic activities and DNA or RNA polymerase fidelity,
although
the link is probably different from that considered originally [12]. It
could
have to do, as I mentioned, with the clearing of the active centre of
the
polymerase from the undesired NTPs. I note in this respect
that hepatitis B virus DNA synthesis
occurs within a core from which PPi and other side products of the
polymerization reaction may have difficulties to escape [28]. The parasitic degradative
reactions may also intervene in vivo, when a polymerase is stalled as a
results of its previous actions, so
the work must be undone [25, 29]. Stalling may also be due to
nucleotide damage
on the template, so it could be that some polymerases carry with them
repair
activities, which, in some cases, are known to generate nucleoside
diphosphates
[30].
+++++++++++++++++++++++
Polymerization/excision
kinetics
I
came out of the previous work on NDP and dNDP forming reactions with
the
feeling that these activities may after all be contaminants, and that
they were
not part of the standard incorporation pathway. So I focused on the
less
controversial topic of the laws of dNMP generation. Most of the work
was
carried out at the beginning, by Franoise Bernardi, with E. coli DNA
polymerase I or, more precisely, its Klenow fragment. This time, the
radioactivity quenching corrections were carefully made. Several
homopolymers
were used as templates, and we followed the ratio of incorporated
versus
excised nucleotides, as the reaction mixture was being depeleted in
substrate.
In at least two systems, the polymerization/excision ratio varied as a
function
of the remaining substrate exactly as expected on the basis of the
next-nucleotide effect . In at least one other system, the ratio was as
expected on the basis of the delayed-escape mechanism.
The
results were briefly given in a review on DNA polymerase accuracy [13],
then
submitted in August 1977 in more complete form, with full mathematics
to the
Journal of Molecular Biology, where it was handled by Sydney Brenner.
There was
a favourable report, but accompanied with requests for experimental
controls.
At that time, Franoise Bernardi was no longer in the laboratory, and
the
controls were carried out by Mohammed Saghi and Mireiille Dorizzi, who
had
taken her succession in the DNA polymerase work. In July 1978, I
attended a
FEBS meeting in Dresden. There, I met Alan Fersht. He told me that he
was a
reviewer, and that he found the manuscript very nice .
However,
our revised version, submitted later in July 1978 received a negative
report
from a reviewer who did not understand our mathematics (he was still
living at the stone age of Michaelis
kinetics).
Fortunately, Sydney Brenner trusted me, and the article was ultimately
published [31].
My
proof of the next-nucleotide effect was complete. It showed how the
misincorporation/excision ratio varied with the concentration of the
correct
nucleotide, when Poly(dC) was the template, dGTP the correct
nucleotide, and
dATP or dCTP the competing incorrect nucleotides (Figures 2 and 3 in
[13], and Fig. 4, Fig. 5 in [31]). The
next-nucleotide effect was subsequently demonstrated with more
sophisticated
experiments, using natural DNA templates and several DNA polymerases
[32-34].
The effect is now considered as obvious in the field of DNA polymerase
mechanisms. Rather than measuring polymerization/excision ratios,
people
usually just measure misincorporations as a function of the next
nucleotide
concentration. This is now being used as a test for detecting the
existence of
an exonuclease activity coupled to the incorporation activity of a
polymerase
(e.g., [35]). The work by Clayton et al. [34] is interesting, it shows
so to
speak how to transform a standard polymerase into an antimutator
polymerase, by
lowering the dNTP levels. Maurice Bessman could have shown this earlier
[36],
but failed to appreciate the next-nucleotide effect, due to an
incorrect
mathematical analysis of his data. The mathematical article [37]
following [36]
introduced though the interesting concept of the random walk of a DNA
polymerase over a template, due to series of forward and backward
steps.The
solution of the simplest kinetic model was given in [9, 31, 38].
Solutions for
more complex cases were given in [39-41].
After
the departure of Franoise
Bernardi to follow her husband in Marseille, the DNA polymerase
kinetics
studies were interrupted until Mireille Dorizzi, an experienced
enzymologist
and Mohammed Saghi, a pre-doctoral fellow from Morocco took the subject
again.
The previous work had provided evidence for two families of kinetic
curves. In
all likelihood, there was a single kinetic mechanism, of which we were
observing different limiting cases. So, it seemed to me important at
this stage
to establish the polymerization/excision kinetics under a wider range
of
substrate concentrations, with the hope that the extended kinetics
would
display the two types of behaviour in different concentration ranges.
Mireille
Dorizzi, on her side, wished to go down to earth and make better
connections
with classical enzymology. So, Saghi and Dorizzi made pH dependence and
temperature dependence studies [42]. They reported that
temperature influences
excision more than inorporation the ratio of the two activities
increasing linearly with temperature. The pH dependency curves possibly
reflected a ionization of the template in one case, and of the
substrate in
another. Although this was not stated in the summary, the
polymerization/excision kinetics were now established with improved
precision.
A type of deviation from the simplest polymerization/excision kinetics,
already
noticed in [31] was furher substantiated. Here there was perhaps a clue
on the
mechanism of the reaction, but it did not fit with any known scheme.
Having
completed and defended his thesis, Mohammed Saghi returned to his
native
country where he teaches molecular biology at the university of Rabat.
The
elucidation of the origin of the
kinetic anomaly occupied me and my co-workers for quite a while. The
work,
described in the next section, was initiated by Catherine Papanicolaou.
The
next nucleotide effect had one important practical consequence :
replication is more accurate at low substrate concentration. The
enzymologists
were taking as an article of faith that all enzymes worked correctly
only when
the substrate concentration was above the Km (whatever Km means). Thus,
all
studies with substrates below Km were discarded as being
unphysiological and
irrelevant. The next-nucleotide effect forced the DNA polymerase
enzymologists
to abandon the stupid prejudice about the necessity of using substrates
above
Km. This was rather important at that time because so much DNA
sequencing work
was done with the PCR reactions, using too high nucleotide
concentrations.
Research scientists are often asked to prove that their work has
practical
applications. In my case, I can thus claim two contributions to
biotechnology : the next-nucleotide effect, and the fast algorithm
for
searching homologies between sequences [43], now known under the name
of BLAST
and often attributed to others (See the web chapter of
Contributions to
bio-informatics ).
Excellent
work has been performed by others on the contributions to DNA
replication
accuracy of incorporation, excision, or pyrophosphorolysis (e.g., [44,
45],
review in [46]). The kinetic mechanisms of DNA polymerases has also
been
dissected with great sophistication using fast kinetic methods, and
taking
advantage of the crystallographic breakthrough (e.g., review in [47]).
I would
like to add here a special mention to a very informative and lucid
review by
Beckman and Loeb [48] touching on polymerization and excision kinetics.
While
elegant, probabilistic mathematical treatments go directly to the point
in
problems of accuracy, the field of accuracy is often spoiled by
irrelevant
stone age enzymological treatments which use Km and Vmax reasonings.
The
Beckman and Loebs review, however, uses correct mathematics. As a
matter of
fact, Robert Beckman detected errors in my J. Mol. Biol. article [31].
One was
a printing confusion : (k3 + k4) in Eq(17)
should
have been (k-3 + k4). More importantly, Robert
Beckman
noted that my statement about the effect of pyrophosphorolysis (page
101, after
Eq. (25)) was incorrect.
The
more general issue there was about polymerization/excision kinetics
that did
not agree with the simplest next nucleotide effect. As observed by
several
authors (see review in [48]) the exonuclease/incorporation ratio
reaches an
asymptote at increasingly high dNTP concentrations, it does not
decrease to
zero as predicted by the simplest next nucleotide effect. The observed
behaviour, on the other hand, was predicted by the
delayed-escape
model which proposes that the exonuclease activity extends over two
distinct
kinetic steps. A similar behaviour can be predicted if there is some
production
of dNMP prior to nucleotide incorporation, according to Hopfields
kinetic
proofreading model PLUS the
classical exonuclease. Kunkel Beckman and Loeb studied the effect of
pyrophospahte [44]. Putting all the evidence together, Beckman and Loeb
concluded [48] :
Although
five proofreading models predict residual proofreading after the next
nucleotide effect is saturated, only one, the modified two-step
proofreading
model (Bernardi et al., 1979), is in accord with the powerful
functional
constraints provided by the study of combination of proofreading probes
(Kunkel
et al., 1986). .
In
this paragraph, Bernardi et al. corresponds here to [31], Kunkel et al.
corresponds here to [44] and the modified two-step proofreading
model is the combined Hopfields kinetic proofreading +
classical
exonuclease model. I would be more cautious on the implications of the
PPi
data. Pyrophosphate may act as a standard product of the incorporation
reaction, and it may also act as a modifier of the enzyme, and it may
also be
the substrate responsible for a delayed-escape effect as proposed by
Kuchta et
al. [16]. To settle matters, it would be useful to add to the PPi experiments, studies which test more
directly possible successive stages in dNMP production. In particular,
it would
be useful to develop a system in which a nucleotide is misincorporated
due to a
transient conformation (e.g., a tautomeric shift) and proofread
efficiently
after switching to the standard conformation.
+++++++++++++++++++++++
Mnemonic
effects and
exonuclease warm-up
In
addition to the kinetic anomaly at high dNTP (non-saturation of the
exonuclease), we had also observed a systematic kinetic anomaly in the
low
substrate concentration range which could not be simply explained by
experimental uncertainties. There seemed to be a deficit in the
exonuclease
function at low concentration of substrate [31, 42].
The
place left vacant in my team by Mohammed Saghi was soon taken by a
pre-doctoral
student, Catherine Papanicolaou. She worked in parallel on two
themes :
(i) energy models for the prediction of RNA secondary structure [49,
50]
see the web chapter contributions to bio-informatics
and
(ii) the DNA polymerase kinetic
anomaly at low substrate.
The
anomaly could be interpreted as reflecting an allosteric effect, but
the DNA
polymerase is a monomeric effect. It had been realized quite early [51,
52]
that apparent allosteric behaviour could in fact be the reflection of
hysteretic effects. The idea is that a monomeric enzyme may exist under
two
conformations, one with high and one with low activity towards the
substrate.
Right after a productive interaction with the substrate, the enzyme is
in the
state in which it is competent for a further interaction with another
molecule
of substrate. However, at low concentration of substrate, as time
elapses, the
enzyme has increasing chances of switching to the state in which it is
incompetent for substrate binding. This is how an apparent
positive
cooperativity is created (e.g., reviews in [53-55]).
In
the case of E. coli DNA polymerase I, all our experiments turned out to
be
consistent with a mnemonic mechanism. More precisely, the crucial
factor was
the interval of time between a dissociation of the DNA polymerase from
the
template, and its subsequent binding to another molecule of template
[14, 56].
After her thesis, Catherine Papanicolaou moved to Lynn Ripleys
laboratory in
the USA, and the work was pursued by the late Philippe Lecomte. If our
interpretation of mnemonic kinetics was correct, a DNA polymerase could
bind to
the template with its exonuclease in a relatively inactive state, and
the
exonuclease would become more and more active as the DNA polymerase
would
progress along the template. We thus undertook the task of determining
how the
nucleotide misincorporations varied according to the number of
elongation
steps. This was a rather high-tech experiment with respect to the state
of the
art in the field. We still worked, it is true, with homopolymers. But
the DNA
chains which were elongated were separated by gel electrophoresis,
allowing us
to determine the misincorporation ratio at each position. Philipe
Lecomte was
doing all the wet biochemistry, and I took the responsibility of the
quantification of the radioactivity in the gel electrophoresis bands.
Here, I
will relate an instructive episode.
In
order to quantify the error levels in each band, we were making
autoradiograms
of the gels. We had bought an expensive densitometer which was scanning
the
autoradiograms through a narrow linear slit, giving a profile of the
density
accross the bands. It was also supposed to make background subtraction
and
provide the integrated values for the denisty of each band. However, I
soon
realized that the numerical figures given by the high tech densitometer
could
not be correct. Instead of separating the bands in terms of slightly
overlapping gaussian curves, the algorithm used by the densitometer
consisted
of going from one local minimum to the next, and take the surface of
the peaks
above the segment joining two consecutive local minima. There followed
a long
and disagreable correspondence with the manufacturer of the
densiometer. There
seemed to be no way to have access to the original (uncorrected) data.
It would
have been an easy task for me to retrieve the correct densities from
the
original data. At last, the manufactuer stated that the method which
they had
implemented to integrate the band densities had been demanded by the
physicians, because this was the way the physicians thought it should
be done
!!! In any event there was nothing they could do at this stage
(but it
seems that in later versions of the densitometer, they proposed as an
option,
the access to the raw data). So I decomposed manually all the density
profiles
into gaussians, and mesaured all surfaces manually on the rolls of
paper coming
out of the densitometer. We thus showed very clearly, with the
Poly(dA).oligo(dT) system that the misincorporation level of A was
rather high
at the +1 position, and it decreased rapidly, from the 1st to the 25th
position
[57]. The work was summarized as follows :
================
A.A
mismatch errors occurring during poly(dA) replication with the Klenow
fragment
of E. coli DNA
polymerase I have
been quantified. The A/T ratio measured for chains extended by 1-25
nucleotides
decreases by a factor of at least 15 from beginning to end. The deduced
true
error rate may decrease by a factor 2.5 at each successive nucleotide
addition.
When ddATP is used instead of dATP, the ddA/T ratio indicates little
variation
of the misincorporation probability with position. Thus, the accuracy
improvement in the first case is due to warm-up of the proofreading
function.
================
On
this account it seemed that the exonuclease activity of E. coli DNA
polymerase
I was nearly inactive following the first incorporation act. However,
strangely
enough, it seemed that a substantial fraction of the initial oligomers
had been
trimmed. This feature was elucidated in a subsequent work. At the end,
we
reached an understanding of the system described as follows in the
summary of
our last paper [58] :
================
Escherichia
coli DNA polymerase I
exists in at
least two distinct kinetic forms. When it binds to a template, the
proofreading
activity is usually switched off. As the enzyme progresses along the
template,
it becomes more and more competent for excision. This phenomenon
introduces a
link between fidelity and processivity. Processivity is best studied
when the
chain length distributions of synthesized polymers are stationary. Even
then,
however, one cannot avoid multiple initiations on a given template by a
same
molecule of the enzyme. When synthesis is initiated with primer lengths
15 or
20, a strange phenomenon is observed. It seems that the polymerase
starts by
hydroyzing the primer down to a length of 7-10 nucleotides, and only
then
starts to add nucleotides. It does so in low-accuracy mode, suggesting
that
while the exonuclease is clearly active, it does not contribute to
proofreading. The warm-up of the proofreading function is therefore
reinterpreted as a switch between two modes of behaviour : a mode
1 of low
accuracy in which the 3
5 exonuclease, while active, is uncoupled from the polymerase and does
not
contribute to proofreading, and a mode 2 of high accuracy in which the
exonuclease is kinetically linked to the polymerase activity.
--------------------------
Our
work was not taken very seriously. In particular, Stephen Benkowic and
co-workers, working on short heteropolymers containing the four
nucleotides
- which looked more physiological
models than our homopolymers - could not reproduce our observations.
Stephen
was kind enough to send us some of his template and we did not observe
with
this template anything like our mnemonic behaviour. Last, we did not
see what
could be the biological rationale for such a property. We wrote that
Allowing the enzyme to excise efficiently only when the cellular
state
permits active incorporation minimizes the risk of accidental
degradation of
DNA , but were not fully convinced by this explanation. Dominique
Job and
his team in Marseille accumulated evidence in favour of a mnemonic
behaviour
for wheat germ RNA polymerase [59]. However, the work did not catch the
attention of the RNA polymerase community.
In
retrospect, I think that our work was quite good, and physiologically
relevant.
Possibly the failure to observe mnemonic effects with Benkovics
template could
be due to the possibility that the template was not sufficiently long
to
facilitate the renaturation of the enzyme, and that the exonuclease was
therefore always working at low efficiency with this oligomer.
Concerning the
biological relevance of an exonuclease warm-up during
elongation,
a possible biological rationale emerged nearly twelve years later [60].
I was
then paying attention to the optimum accuracy of DNA repair enzymes,
and found,
quite naturally, that the shorter a repair patch, the higher the
tolerable
level of repair errors. So an enzme could start at a rather high level
of
errors, and switch to lower levels further along on the repair patch. I
also
explained why some inaccuracy was useful in the case of DNA repair [60].
On
the whole, I find that this DNA polymerase work was not a loss of time.
In some
aspects, it was not as high-tech as other works (we worked on
homopolymers, we
used E. coli Pol I which is now thought to be a repair enzyme, and we
did not
perform fast kinetic experiments). It was however for some other
aspects at a
quite good technical level (the separation, for each time in the
reaction of
the products of the reaction, either by thin-layer chromatography, to
quantify
the dNMPs, the dNDPs and the remaining dNTPS, or later, the
determination of
misincorporation levels position by position using sequencing gels).
The
article in BBA with Philippe Lecomte [58] was my last published
experimental work
in molecular biology. Philippe Lecomte made some further experiments
with a
periodic polymer, Poly(T-C-A)30, raising progressively the
pyrophosphate concentration to act selectively on a single type of base
pair
then on another one. I find in my notes from that time that at moderate
PPi, chain extension paused after
the C sites as expected. At higher PPi, there was no polymerization,
but
polymerization occurred again at still higher PPi, for reasons which
were not
elucidated (or perhaps this result was an artefact).
+++++++++++++++++++++++++++++++++++++++++++++++
REFERENCES
[1] Brutlag, D. and Kornberg, A. (1972)
Enzymatic
synthesis of deoxyribonucleic acid. XXXVI. A proofreading function for
the 3 5 exonuclease activity in
deoxyribonucleic acid
polymerases. J. Biol. Chem.
247, 241-248.
[2] Muzyczka, N., Poland, R.L. and
Bessman, M
J. (1972) Studies on the biochemical basis of spontaneous mutation. 1.
A
comparison of the deoxribonucleic acid polymerases of mutator,
antimutator and
wild type strains of bacteriophage T4. J. Biol. Chem.
247, 7116-7122.
[3] Ninio, J. (1974) A semi-quantitative
treatment of
missense and nonsense suppression in the strA and ram ribosomal mutants
of Escherichia
coli . Evaluation of some
molecular
parameters of translation in vivo.
J. Mol. Biol. 84, 297-313.
[4] Englund, P. T., Huberman, J. A.,
Jovin, T. M. and
Kornberg, A. (1969) Enzymatic synthesis of deoxyribonucleic acid. XXX.
Binding
of triphosphates to deoxyribonucleic acid polymerase. J. Biol. Chem.
244,
3038-3044.
[5] Josse, J., Kaiser, A. D. and Kornberg,
A. (1961)
Enzymatic synthesis of deoxyribonucleic acid. VIII. Frequencies of
nearest
neighbour base sequences in deoxyribonucleic acid. J. Biol. Chem. 236,
864-875.
[6] Hershfield, M.S. (1973) On the role of
deoxyribonucleic acid polymerase in determining mutation rates. J.
Biol. Chem.
248, 1417-1423.
[7] Hall, Z.W. and Lehman, I.R. (1968) An in
vitro transversion by a
mutationally altered T4-induced
DNA polymerase. J. Mol. Biol. 36, 321-333.
[8] Howard, B. D. and
Tessman, I. (1964) Identification of the altered bases
in mutated single-stranded DNA. II. In vivo mutagenesis by 5-bromodeoxyuridine and
2-aminopurine.
J. Mol. Biol. 9, 364-371.
[9] Ninio, J.(1975) Kinetic amplification
of enzyme
discrimination. Biochimie 57, 587-595.
[10]
Hopfield, J.J. (1974) Kinetic proofreading : A new mechanism for
reducing
errors in biosynthetic processes requiring high specificity. Proc. Nat.
Acad.
Sci. USA 71, 4135-4139.
[11] Ninio, J. (2006) Multiple stages in
codon-anticodon recognition : double-trigger mehanisms and
geometric
constraints. Bochimie 88, 963-992.
[12] Ninio, J., Bernardi, F., Brun, G.,
Assairi, L.,
Lauber, M. and Chapeville, F. (1975) On the mechanism of nucleotide
incorporation into DNA and RNA. FEBS Letters 57, 139-144.
[13] Bernardi, F. and Ninio, J. (1978) The
accuracy
of DNA replication. Biochimie 60, 1083-1095.
[14] Papanicolaou, C., Dorizzi, M. and
Ninio, J.
(1984) A memory effect in DNA replication. Biochimie 66, 43-48.
[15] Joyce, C. M. and Steitz, T. A. (1987)
DNA
polymerase I : from crystal structure to function via genetics.
Trends in
Biochem. Sci. 12, 288-292.
[16] Kuchta, R. D., Benkovic,
P. and Benkovic, S. J. (1988) Kinetic mechanism
whereby DNA polymerase I (Klenow) replicates DNA with high fidelity.
Biochemistry 27, 6716-6725.
[17]
Ninio, J. (1991) Transient mutators: a semiquantitative analysis of the
influence of translation and transcription errors on mutation rates.
Genetics 129,
957-962.
[18] Scheuermann, R. H. and Echols, H.
(1984) A
separate editing exonuclease for DNA replication : the e subunit of Escherichia coli DNA polymerase III holoenzyme. Proc. Nat.
Acad. Sci.
USA 81, 7747-7751.
[19] Ninio, J. (1986) Kinetic and
probabilistic
thinking in accuracy. In Accuracy in Molecular Processes (Kirkwood. T.B.L., Rosenberger, R. and
Galas, D.J.,
eds) Chapman & Hall, London, pp. 291-328.
[20] Blank, A.,
Gallant, J.A., Burgess, R. R. and Loeb, L. A. (1986) An RNA
polymerase
mutant with reduced accuracy of chain elongation. Biochemistry 25,
5920-5928.
[21] Libby, R. T., Nelson, J. L., Calvo,
J. M. and
Gallant, J. A. (1989) Transcriptional proofreading in Escherichia
coli. EMBO J. 8, 3153-3158.
[22] Libby, R. T. and Gallant, J. A.
(1991) The role
of RNA polymerase in transcriprional fidelity. Molecular Microbiology
5,
999-1004.
[23] Libby, R. T. and Gallant, J. A.
(1994)
Phosphorolytic error correction during transcription. Molecular
Microbiology
12, 121-129.
[24]
Volloch, V.Z., Rits, S. and Tumerman, L. (1979)
Pyrophosphate-condensing
activity linked to nucleic acid synthesis. Nucleic Acid Res. 6,
1521-1534.
[25 ]
Volloch, V.Z., Rits, S. and Tumerman, L. (1979) A possible mechanism
for the
correction of transcription errors. Nucleic Acids Res. 6, 1535-1546.
[26] Hinrichsen, A. I., Ortner, I. M. and
Hartmann,
G. R. (1985) Synthesis of dinucleoside tetraphosphates by RNA
polymerase B (II)
from calf thymus. FEBS Lett. 193, 199-202.
[27] Butzow, J. J., Garland, C. Van Lee,
L. and
Eichhorn, G. L. (1997) Specificity of an RNA polymerase-associated
NTPase.
Biochemistry 36, 14794-14798.
[28] Urban, S., Urban, S., Fischer, K. P.
and Tyrell,
D. L. (2001) Efficient pyrophosphorolysis by a hepatitis B virus
polymerase may
be a primer-unblocking mechanism. Proc. Nat. Acad. Sci. USA 98,
4984-4989.
[29] Rozovskaya, T. A., Chenchik, A. A.
and
Beabealashvilli, R. S. (1982) Processive pyrophosphorolysis of RNA by Escherichia
coli RNA polymerase. FEBS
Lett. 137,
100-104.
[30] Postel, E. H. and Abramczyk, B. M.
(2003) Escherichia
coli diphosphate kinase is
a
uracil-processing DNA repair nuclease. Proc. Nat. Acad. Sci. USA 100,
13247-13252.
[31] Bernardi, F., Saghi, M., Dorizzi, M.
and Ninio,
J. (1979) A new approach to DNA polymerase kinetics. J. Mol. Biol. 129,
93-112.
[32] Fersht, A. R. (1979) Fidelity of
replication of
phage X174 DNA by DNA polymerase III holoenzyme : Spontaneous
mutation by
misincorporation. Proc. Nat. Acad. Sci. USA 76, 4946-4950.
[33] Clayton, L. K., Goodman, M. F.,
Branscomb, E. W.
and Galas, D. J. (1979) Error induction and correction by mutant and
wildt type
T4 DNA polymerases. Kinetic error discrimination mechanisms. J. Biol.
Chem.
254, 1902-1912.
[34] Kunkel, T. A., Schaaper, R. M.,
Beckman, R. A.
and Loeb, L. A. (1981) On the fidelity of DNA replication. Effect of
the next
nucleotide on proofreading. J. Biol. Chem. 256, 9883-9889.
[35] Phear, G., Nalbartoglou, J. and
Meuth, M. (1987)
Next-nucleotide effects in mutations driven by DNA precursor pool
imbalances at
the aprt locus of Chinese hamster ovary cells. Proc. Nat. Acad. Sci.
USA 84,
4450-4454.
[36] Bessman, M.J., Myzyczka, N., Goodman,
M.F.
Schnaar, R.L. (1974) Studies on the biochemical basis of spontaneous
mutation
II. The incorporation of a base and its analogue into DNA by wild-type,
mutator
and antimutator DNA polymerases. J. Mol. Biol. 88, 409-421 ou 422.
[37] Goodman, M.F., Gore, W.C., Muzyczka,
N. and
Bessman, M.J. (1974) Studies on the biochemical basis of spontaneous
mutation.
III. Rate model for DNA-polymerase-effected nucleotide misincorporation. J. Mol. Biol. 88, 423-435.
[38] Galas, D.J. and Branscomb, E.W
.(1978) Enzymatic
determinants of DNA polymerase accuracy. J. Mol. Biol. 124, 653-687.
[39] Herbomel, P. and Ninio, J. (1980)
Fidlit d'une
raction de polymrisation selon la proximit de l'quilibre.
Comptes-Rendus
Acad. Sci. Paris, Srie D, 291, 881-884.
[40] Malygin, E.G. and Yashina, L.N.
(1980) Kinetic
description of the error-correcting mechanism of bifunctional DNA
polymerases
(in Russian). Doklad. Akad. Nauk. SSSR 250, 246-250.
[41] Durup, J. (1982) On the relation
between error
rates in DNA replication and elementary chemical rate constants. J.
Theoret.
Biol. 94, 607-632.
[42] Saghi, M. and Dorizzi, M. (1982)
Polymerization/excision kinetics of Escherichia coli DNA polymerase I. Stability in kinetic
behaviour and
variation of the rate constants with temperature and pH. Eur. J.
Biochem. 123,
191-199.
[43] Dumas, J.-P. and Ninio, J. (1982)
Efficient
algorithms for folding and comparing nucleic acid sequences. Nucleic
Acids Res.
10, 197-206.
[44] Kunkel, T.A., Beckman, R. A. and
Loeb, L. A.
(1986) On the fidelity of DNA synthesis. Pyrophosphate-induced
misincorporation
allows detection of two proofreading mechanisms. J. Biol. Chem. 261,
13610-13616.
[45] Schaaper, R. M. (1993) Base
selection, proofreading
and mismatch repair during DNA replication in Escherichia coli. J. Biol. Chem. 268, 23762-23765.
[46] Kunkel, T. A. and Bebenek, K. (2000)
DNA
replication fidelity. Annual Rev. Biochem. 69, 497-529.
[47] Johnson, K. A. (1993) Conformational
coupling in
DNA-polymerase fidelity. Annu. Rev. Biochem. 62, 685-713.
[48] Beckman, R.A. and Loeb, L.A. (1993) Multi-stage proofreading in DNA
replication. Quarterly Reviews of Biophysics 26, 225-311.
[49] Papanicolaou, C., Gouy, M. and Ninio,
J. (1984)
An energy model that predicts the correct folding of both the tRNA and
the 5S
RNA molecules. Nucleic Acids Res. 12, 31-44.
[50] Gouy, M., Marlire, P., Papanicolaou,
C. and
Ninio, J. (1985) Prediction des structures secondaires dans les acides
nuclques: aspects algorithmiques et physiques. Biochimie 67, 523-531.
[51] Witzel, H. (1967) Zur Interpretation
kinetischer
Daten bei der Reaktion der Pankreas-Ribonuclease. Hoppe-Sylers Z.
Physiol.
Chem. 348, 1249-XXXX.
[52] Rabin, B. R. (1967) Co-operative
effects in enzyme
catalysis a possible model based on substrate-induced conformation
isomerization. Biochem. J. 102, 22C 23C.
[53] Neet, K. R. and Ainslie, G. R. Jr
(1980)
Hysteretic enzymes. Methods in Enzymology 64B, 192-226.
[54] Ricard J, Meunier J. C. and Buc, J.
(1974)
Regulatory behaviour of monomeric enzymes. 1. Mnemonical enzyme
concept. Eur.
J. Biochem. 49, 195-208.
[55] Cornish-Bowden, A. and Cardenas, M.L.
(1987)
Co-operativity in monomeric enzymes. J. Theoret. Biol. 124, 1-23.
[56] Papanicolaou, C., Lecomte, P. and
Ninio, J.
(1986) Mnemonic aspects of E. coli
DNA polymerase I. Interaction with one template influences the next
interaction
with another template. J. Mol. Biol. 189, 435-448.
[57] Lecomte, P.J. and Ninio, J. (1988).
Nucleotide
excision by E. coli DNA polymerase I in proofreading and
non-proofreading modes. Biochim. et Biophys. Acta 951, 255-260.
[58] Lecomte, P.J. and Ninio, J. (1987)
Variations
with position of replication errors due to exonuclease warm-up. FEBS
Lett. 221,
194-198.
[59] Job, D., Souli, J. M., Job, C. and
Shire, D.
(1988) Potential memory and hysteretic effects in transcription. J.
Theoret.
Biol. 134, 273-289.
[60] Ninio, J. (2000) Illusory defects and
mimatches
- or why must DNA repair always be (slightly) error-prone? BioEssays,
22,
396-401.