Table Of ContentAdvances in the Biosciences
Editor: G. Raspe
Associate Editor: S. Bernhard
Technical Assistance:
H. Schmidt
The Schering Symposia and Workshop Conferences
are conducted and sponsored by
Schering AG, 1 Berlin 65, Müllerstraße 170
Advances in the Biosciences 8
Workshop on
Mechanisms and Prospects
of Genetic Exchange
Berlin, December 11 to 13,1971
Editor: Gerhard Raspe
Associate Editor: S. Bernhard
Editorial Board: Peter Hans Hofschneider
Hilary Koprowski
Pergamon Press · Vieweg
Oxford · Edinburgh · New York · Toronto · Sidney · Braunschweig
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ISBN 3 528 076909 (Vieweg)
1972
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Copyright ©1972 by Friedr. Vieweg + Sohn GmbH, Verlag, Braunschweig
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Opening Address
Heinz Gibian
Forschungsleitung Pharma der Schering AG, Berlin, Germany
Dr. Raspe, who has unfortunately fallen ill, has asked me to extend a very warm
welcome to you on behalf of Schering AG in the opening of this eighth workshop.
When the very first conference in this series was held in 1967 on the initiative of
Dr. Raspe, no one could have foreseen that we would meet today in a workshop on
"Mechanisms and Prospects of Genetic Exchange". The reason for this becomes
immediately obvious when I mention the subject of our first meeting "Symposium
on Endocrinology". That was, of course, a subject which was very closely connected
with our own research; the same was true for the following workshops, at which
we had our own contributions to make. This time, however, we are merely listeners.
Despite this, the seeds had already been sown for the selection of today's subject.
There are areas of science in the process of an exciting upsurge, which keep us ex
tremely alert intellectually, but which, nevertheless, have no place to call their own
since, in the choice of methods, they are interdisciplinary. Perhaps, to quote Günter
Stent, it is more a case of the representatives of such areas who see themselves in a
"romantic phase", to which institutionalization would put an end.
However that may be, from time to time it is necessary for the development of such
areas that the right people from the various disciplines come together at the right
place. In the New World this requirement is met admirably by the Cold Spring
Harbor meetings, Gordon conferences, and similar meetings. Here, in the Old World,
much is left to be desired in this direction; the Schering Workshops are intended to
be a contribution to this problem.
New developments should be encouraged in the discussion between the disciplines.
Outsiders should be informed about them for it is very possible that they, in
particular, will have unexpected possibilities for future research. We attach the
greatest importance to the presence of young colleagues here. Every invited speaker
1 Advances 8
10
was given the opportunity of naming, as a participant, a younger scientist from
whatever country he wished. We are trying to realize this principle for each work
shop. As with everything else concerned with this workshop, there are no hard and
fast rules; this also applied to the selection of topics.
The fascinating subject of this conference is the result of a discussion between
Hilary Koprowski and Peter Hans Hofschneider. We agreed to their brilliant proposal
very quickly. I should think that even the initiators of this workshop were somewhat
surprised — and a bit worried — about this speed as it meant a special responsibility
for them in view of the short time available for preparations. The same is true for
Silke Bernhard, whom I should like to thank very much for her magnificent efforts
in organizing this workshop.
My first wish for the next few days, during which molecularbiologists, geneticists,
cell biologists, biophysicists, and immunologists will be communicating with each
other, is that new bridges will be built during the official discussions, over a glass of
wine, or on a visit to one of the charming Berlin restaurants. My second wish will
then become true all by itself: We will obtain a competent intermediate balance
within a field which is of interest not only to scientists, but also to the public.
Since the memorable conference "Man and His Future" in London, speculations,
fears, and Utopia about a genetic dictatorship of mankind through science have not
ceased. It would be in everyone's interest if the basis to the facts could be presented.
With this in mind, my colleagues and I are very happy to be your hosts.
I should like to thank all of you very cordially for having come here; my special
gratitude goes to the lecturers without whom this workshop would not have been
possible.
Advances in the Biosciences 8
Introduction to Session I
Robert L. Sinsheimer
California Institute of Technology, Division of Biology, Pasadena, California
When one starts to think seriously about the possibilities for the development of
genetic therapies or genetic change, one quickly confronts a set of problems that
has to do with the insertion or the deletion or the transposition of pieces of genetic
material - DNA pieces which might range from a few nucleotides in length to
several genes — into an existing genome. Now in a formal sense, models for the solu
tion of such questions clearly exist in nature in the processes of genetic recombina
tion, of provirus and episomal insertion, of genetic inversion, in heterochromatic
condensation, and in the postulated gene expansion and contraction. As yet, of
course, we know very little of the molecular events involved in such action.
At least two general mechanisms of genetic recombination appear to have been
developed. One seems to require at least a fair degree of genetic homology between
the recombining DNAs; it seems to be favoured by the presence of nicks in DNA,
and to be mediated in bacteria at least by the so-called rec genes. The second mech
anism for which provirus insertion is a good model does not appear to require
any extensive homology between the insertant and the insertee, and appears to
be mediated by enzymes specifically evolved with recognition capacities for that
purpose.
Variants of these mechanisms can certainly be imagined and most likely exist. And,
indeed, wholly distinct processes of recombination may await discovery. 1 am not
at all sure that the events that lead to recombination during meiosis — the formation
of the synaptinemal complex and so on - bear any simple relationship to those
events that 1 have already mentioned.
At the molecular level, these events clearly involve processes of molecular recogni
tion of undefined extent, processes of chain scission, chain extension, and chain
ligation for which we have putative model catalysts. They also very likely involve
processes of chain initiation and chain termination which are still obscure. The
papers that will be presented today will be concerned with our understanding of
the molecular and genetic events underlying processes of gene interaction and
gene exchange.
Advances in the Biosciences 8
Enzymology of Genetic Recombination
Charles M. Radding and Era Cassuto
Departments of Medicine and Molecular Biophysics and Biochemistry, Yale University School
of Medicine, New Haven, Conn. 06510, USA
Summary: Studies of genetic recombination in prokaryotes have shown (1) that recombination
occurs by breakage and reunion of DNA, sometimes, but not always, associated with new DNA
synthesis, and (2) that the parental contributions to a recombinant molecule are commonly
joined by a short heteroduplex or hybrid region. In the past few years, some of the enzymes
involved in recombination in prokaryotes have been identified, such as the exonuclease made
by bacteriophage λ. Recent studies of λ exonuclease make it possible to rationalize most of the
properties of the enzyme in terms of its role in producing a perfect heteroduplex joint between
homologous molecules of DNA. λ exonuclease cleaves 5' mononucleotides from the 5' end of
native DNA in a processive fashion, extensively degrading any molecule of DNA before detach
ing and attacking another molecule of substrate. The latter property suggests that some control
prevents the enzyme from playing an exclusively degradative role. 5' phosphoryl termini located
at gaps in one strand of duplex DNA are resistant to the enzyme. Although 5' phosphoryl termini
at nicks are even more resistant, the enzyme appears to bind weakly at such sites. The significance
of these properties may be seen in the enzyme's action at the site of a redundant single stranded
branch, such as one might expect to find at a joint between two fragments of DNA. A redundant
strand is assimilated into the helix, behind λ exonuclease, as the enzyme processively degrades
the homologous helical strand. The enzyme recognizes the presence of the redundant strand
both for initiation and termination of hydrolysis. Removal of the redundant single strand by
the prior action of exonuclease I blocks the action of λ exonuclease on the helical strand. More
over, when a redundant strand has been completely assimilated through the action of λ exonuclease,
the enzyme stops at the precise point which permits the interrupted polynucleotide strand to
be sealed by polynucleotide ligase. The sequential action of λ exonuclease and polynucleotide
ligase on redundant joint molecules of λ DNA produces intact polynucleotide strands that are
biologically active. Several models have been suggested to relate the assimilation of single strands
to the genetic recombination of λ and possibly to recombination in other systems as well.
Molecules of DNA with double-stranded branches have also been synthesized to test one of the
Manuscript received: 13 December 1971
14 Ch. M. Radding and E. Cassuto
models. The models suggest that λ exonuclease may catalyze a concerted reaction that (1)
exposes complementary nucleotide sequences, (2) forms or extends the heteroduplex region,
and (3) eliminates redundant branches, precisely restoring a duplex structure that can be sealed
covalently by polynucleotide ligase. The λ enzyme, and similar exonucleases, might drive other
wise reversible interactions of a single strand with a recipient duplex, including certain kinds of
interactions between two molecules of double-stranded DNA.
Introduction
Genetic recombination is the set of processes that leads to new linkage relationships.
By greatly accelerating genetic permutations, recombination has probably played
an important role in evolution. For the present, recombination is of interest as an
aspect of DNA metabolism in which complicated and poorly understood relation
ships exist among replication, repair, and recombination. In the future, our under
standing of genetic recombination may influence our ability to deal with such
medically important phenomena as resistance transfer factors, carcinogenesis, and
gene therapy.
There are three possible objects to study in recombination: the progeny, the inter
mediates, or the enzymes. In the past few years, explorations of recombination
deficient mutants in prokaryotes have strongly implicated certain enzymes in
recombination, thus making it possible to approach the enzymology with greater
assurance [9, 33]. One such enzyme, the exonuclease made by bacteriophage λ,
is the product of a gene that is essential for recombination of the phage [25, 31, 34].
Recent studies of λ exonuclease make it possible to rationalize most of the properties
of the enzyme in terms of its role in perfectly splicing homologous portions of DNA.
These experiments will be summarized after a brief digression on the features of
recombination in prokaryotes that are particularly relevant to an enzymic analysis.
Types
Most recombination can occur more or less anywhere along the length of two
homologous genophores, and is called general recombination. A special type of
recombination that occurs only at a restricted number of specific sites is called
site specific recombination [14]. Still a third type, sometimes called unusual or
illegitimate recombination [10], occurs between genophores which may have localized
regions of homology, but which are carrying distinctly different genetic messages.
In this paper we shall deal only with general recombination.
Reciprocity
An individual act of recombination between distant markers (AM X am) may give
rise to reciprocal products (both Am and aM) or nonreciprocal products (either
Am or aM). Different recombination systems appear to be either largely reciprocal
or largely nonreciprocal [4, 23, 33].
Enzymology of Genetic Recombination 15
Material exchange and DNA synthesis
Studies of the progeny of recombination show that the parents usually contribute
most of the material of which the recombinant molecule is made. Newly synthesized
DNA is detectable in some recombinant molecules but not in others [22, 23, 36, 37,
45]. Three possible relationships between DNA synthesis and recombination may
be imagined: (1) New DNA synthesis repairs gaps in certain intermediate structures.
(2) Replication, by producing interruptions and branches in DNA, provides the sub
strate for recombination which then proceeds by mechanisms that are independent
of replication. (3) New DNA synthesis is an intrinsic part of the mechanism of
recombination (see Discussion).
Precision
Nucleotides are rarely gained or lost in recombination. In a cross represented by
ABCD X abed, all four letters are represented in recombinant progeny, for example
ABcd. Repetitions or deletions such as ABB cd or Acd are rare. This precision is
accomplished in general recombination through the pairing of homologous bases,
but the details ofthat process are obscure. Does recognition of the sequence homology
of two molecules of DNA occur before or after the breakage of one or both strands
of each parent? The two general possibilities might be represented as break and join
vs. join and break. The work of Alberts et al. has provided new insights into the
biological mechanisms for making and breaking hydrogen bonds [1].
Heterozygosis
In prokaryotes, the parental contributions of DNA are usually joined by a short
region in which each parent contributes one strand of this duplex DNA. If mutations
are present in the heteroduplex region, heterozygosity may be observed [23].
Heterozygosity based on a heteroduplex junction between the two parental arms
of a recombinant molecule is intimately related to the basic mechanism of recombina
tion and to its precision. Studies of intermediates in recombination have revealed a
stage at which the parental contributions are united only by hydrogen bonds which
are presumably located in the heteroduplex region [17, 39]. Such intermediates are
called joint molecules [39]. An unanticipated way in which heteroduplex regions
may be generated is revealed by the studies on λ exonuclease (see below).
Multiple exchanges
Genetic exchanges tend to be clustered; the site of an exchange is frequently the
site of nearby exchanges [33]. This may turn out to be the most difficult property
of recombination to analyze by an enzymologic approach, unless clustering is shown
to result from the excision and repair of mismatched bases in the heteroduplex
junction [30,40, 45].
16 Ch. M. Radding and E. Cassuto
Properties of λ exonuclease
To find out what λ exonuclease does in genetic recombination, we began to reevaluate
the properties of the enzyme with particular attention to its possible action at
internal sites in DNA such as nicks, gaps, and branches. Some of the elementary
properties of λ exonuclease are outlined in Fig. 1. The observation that the enzyme
acts processively, extensively degrading any molecule of DNA on which it starts,
led to the inference that some control exists which prevents the enzyme from
playing an exclusively degradative role [6, 26]. The specificity of the enzyme for
native DNA suggested that the enzyme did not act by degrading redundant single-
stranded branches. In spite of the specificity of λ exonuclease for native DNA,
binding of the enzyme to denatured DNA has also been observed [28]. Examination
1. (a) highly specific for native DNA [20],
(b) but binds to denatured DNA [28]
2. processively cleaves 5' mononucleotides from the 5' phosphoryl end of native DNA [6, 20]
3. (a) does not initiate digestion at a 5' phosphoryl terminus located at a nick [6, 21],
(b) but binds to nicks [26]
4. does not readily initiate digestion at a 5' phosphoryl terminus located at a gap [6, 12, 21,
27]
Fig. 1. Properties of λ exonuclease.
of the action of λ exonuclease at internal sites, first nicks and then gaps, gave negative
results [6], but another apparent paradox was noted. Although the enzyme shows
no tendency to act at nicks, it appears to bind at such sites [26]. On the basis of
these properties, we made the hypothesis shown in Fig. 2 [8]. According to this
notion, λ exonuclease degrades native DNA at the site of a single-stranded branch
(called a redundant joint) making way for assimilation of the branch into the helix.
When the redundant strand has been completely assimilated, further digestion by
the enzyme stops, leaving a nick that can be sealed by polynucleotide ligase. The
hypothesis rationalizes the properties subsumed under 1—3 in Fig. 1.
Single-strand assimilation
Two different substrates have been synthesized to test the action of λ exonuclease
on redundant joints (Figs. 3, 4). In each case, the normal ends of λ DNA were pro
tected and the only 5' terminus that was potentially available to the enzyme was
the one located at a joint in the middle of the DNA molecule. Protection of the
ends was achieved in one of two ways: (1) The complementary ends of λ DNA were
annealed to form either circles or polymers. (2) The 5' single-stranded termini of λ
DNA were dephosphorylated by E. coli alkaline phosphatase. This treatment reduced
