R698
Dispatch
Chromosome cohesion: A polymerase for chromosome bridges
Frank Uhlmann
How do cells ensure that sister chromatids produced
during DNA replication stay connected with each other
until their separation in anaphase? New insight is
provided by the discovery of DNA polymerase κ, which
has been found to be required for building the
connections between sister chromatids.
Address: Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields,
London WC2A 3PX, UK.
E-mail: [email protected]
Current Biology 2000, 10:R698–R700
0960-9822/00/$ – see front matter
© 2000 Elsevier Science Ltd. All rights reserved.
All the genetic information in a cell is duplicated once in
each cell cycle to be distributed equally into daughter
cells. The faithful execution of this process depends not
only on producing exact copies of each chromosome
during DNA replication, but also on the exact distribution
of these copies in mitosis. As they emerge during DNA
replication, the sister chromatids must become linked to
each other. This cohesion ensures that the sister chromatids
stay together later in mitosis when they are aligned pairwise on the metaphase plate. How the requisite cohesion
between sister chromatids is established during DNA
replication is still a mystery. This is why the recent
discovery of DNA polymerase κ is important [1]. This
unexpected enzyme has been found to be part of the
machinery that links sister DNA strands. This finding will
give new impetus to the current search for the molecular
basis of sister chromatid cohesion.
To understand the significance of this discovery, let me
first describe what is known about how sister chromatids are
held together. A premise of recent work has been that there
are specialised proteins that act as physical bridges between
sister chromatids. Genetic and biochemical approaches
together identified a chromosomal protein complex, called
cohesin, that is an excellent candidate for being a component of such bridges (reviewed in [2,3]). In budding yeast,
cohesin is required from S phase to metaphase to prevent
sister chromatids from premature dissociation. The pairing
of chromosomes by cohesin allows the bipolar attachment
of the sister chromatids to the mitotic spindle [4]. Cohesin
is then needed to withstand the pulling forces exerted by
the spindle that tries to separate sister chromatids from each
other. It is only in anaphase that the cell actively destroys
the cohesin complex to let the sister chromatids separate.
Cohesin consists of at least four protein subunits (see [2,3]
for details and references). Two of these subunits, Smc1
and Smc3, belong to the ‘structural maintenance of
chromosomes’ (hence Smc) family of proteins, members
of which are involved in different aspects of chromosome
dynamics. Smc proteins form large V-shaped dimers of
α-helical coiled-coils with a flexible hinge in the middle
and head domains at both ends (Figure 1). These heads
have the potential to bind to DNA and might therefore
form the connections between cohesin and the chromosome. The heads also contain an ATPase activity that
might regulate higher-order interactions between Smc
proteins, and might also regulate their DNA-binding
activity [5–7]. The other two subunits of the complex are
the ‘sister chromatid cohesion’ proteins Scc1 (also known
as Mcd1) and Scc3. All of the subunits are essential to
form a functional complex. It is the Scc1 subunit that is
then proteolytically cut to destroy the cohesin complex at
the onset of anaphase. Cohesion between DNA strands
mediated by the cohesin complex has not yet been directly
observed. Nevertheless, a first working hypothesis for how
cohesion between sister chromatids might be accomplished has been put forward.
Much less clear is how cohesion between the newly
synthesised sister chromatids is established during DNA
replication. Well before the onset of DNA synthesis,
cohesin pre-binds to the unreplicated chromosomes
(Figure 1). This binding itself has to be helped by an
additional protein complex, made up of the proteins Scc2
and Scc4 [8]. This ‘cohesin loading factor’ might be a
protein kinase that enables cohesin to bind to chromosomes by phosphorylating one of its subunits (S. Jones and
J. Sgouros, personal communication). Components of the
cohesin complex itself become essential during the
replication process. This supports the idea that cohesion is
established during DNA replication by the same proteins
that then hold the sister chromatids together throughout
G2 phase and mitosis. How the pre-bound cohesin
complex is turned into a physical link between sister DNA
molecules during replication, however, is one of the
crucial unanswered questions.
One protein that is required for exactly this process of
building connections between sister chromatids from the
pre-bound cohesin complex has been identified by genetic
analyses in budding yeast, and is called Eco1 or Ctf7. The
biochemical function of Eco1, however, is still mysterious.
Interestingly, the fission yeast homologue of Eco1, called
Eso1, is a much larger protein: it has an Eco1-like carboxyterminal region connected to an amino-terminal region
similar in sequence to DNA polymerase η. Polymerase η in
turn is a DNA repair enzyme that is able to perform DNA
Dispatch
R699
Figure 1
The steps involved in establishing sister
chromatid cohesion during DNA replication.
The cohesin complex — consisting of the four
types of subunit Smc1, Smc3, Scc1 and
Scc3 — is loaded onto DNA with the help of
the Scc2–Scc4 complex. During passage of
the replication fork, cohesin is used to build
connections between the duplicated sister
chromatids. Eco1 is required for this
transition, and a recently discovered DNA
polymerase, polymerase κ [1], has also been
found to be crucially involved.
Scc2
Scc4
Polymerase κ
Eco1
Current Biology
Scc1/3
Smc1/3
synthesis across DNA lesions, such as thymine dimers,
where the polymerases at the replication fork fail [9]. The
DNA repair function and the cohesion establishment function of Eso1 seem to be independent and separable [10].
Still it is tempting to ask whether there is mechanistic connection that brings together a DNA repair polymerase and
a cohesion establishment factor in one polypeptide?
This is where the discovery of polymerase κ comes in. Polymerase κ was previously known as Trf4 in budding yeast.
Trf4 was initially identified as a topoisomerase I-related
activity in a screen for mutations that are lethal in the
absence of the non-essential topoisomerase I [11]. Trf4
itself is not, however, a topoisomerase, and unexpectedly
trf4 mutant cells turned out to be defective in sister chromatid cohesion [1]. The observed cohesion defect is not
quite as dramatic as for mutants in cohesin subunits, but this
could be because there is a close homolog to Trf4, Trf5, that
performs a partly redundant function [12]. The cohesion
defect in trf4 mutant cells could indeed also be the cause for
their previously described hypersensitivity to DNA damage
and defective chromosome condensation [13,14].
The primary defect of trf4 mutant cells only became clear
after sequence motif analysis showed that Trf4 belongs to
the DNA polymerase β superfamily [15]. As well as DNA
polymerase β, a polymerase involved in short-patch baseexcision repair, this superfamily includes a number of
other nucleotidyl transferases, such as poly-A polymerase
which adds the poly-A tails to messenger RNAs. Detailed
biochemical analysis of recombinant Trf4 protein showed
that it indeed is a bona fide DNA polymerase — that is, it
uses a primer on a template DNA strand to synthesise the
complementary DNA strand [1]. In the list of known
DNA polymerases, which follow the Greek alphabet, Trf4
is now called DNA polymerase κ.
Polymerase κ has activities reminiscent of DNA polymerase
β — it polymerises DNA one nucleotide at a time and
Replication
fork
Polymerase κ
Eco1
dissociates from the template after each added nucleotide.
This mode of action is called distributive, and becomes
experimentally most obvious if an excess of primed sites
are offered in a reaction [1]. It is not clear whether polymerase κ, like the main replicative DNA polymerases δ
and ε, might make use of the processivity factor PCNA to
become more processive. PCNA forms a ring around DNA
that acts as a tether to which polymerases can hold on to
[16]. Although entirely speculative, this could explain a
possible involvement of PCNA in the process of sister
chromatid cohesion, suggested by the finding that PCNA
interacts genetically with mutations in Eco1/Ctf7 [17].
What then is the role of polymerase κ in the cell?
Polymerase κ alone is not essential for DNA replication, as
it seems to share its function with Trf5. If both proteins
are inactivated, however, DNA replication stops at an
early stage and proceeds very slowly, if at all. This makes
the two isoforms of polymerase κ together essential [1].
The failure of cells to replicate in the absence of polymerase
κ function shows that they perform an important step
during chromosomal DNA replication. Is it just the polymerase activity of polymerase κ that is needed for DNA
replication, or might cooperation with topoisomerase I be
involved? Another possibility is that a DNA damage signal
might be produced as a consequence of lacking polymerase κ activity, and that could cause the cell-cycle
machinery to delay progression through S phase.
Intuitively, one might expect polymerase κ to perform its
essential function during the process of DNA replication.
But actually polymerase κ is required even in metaphasearrested cells for the maintenance of cohesion between
sister chromatids [1]. This is a tricky experiment, however,
and the observed effect was mild. But maybe there is
more to Trf4 function than the polymerase κ activity? To
address this, point mutations were constructed in polymerase κ that are thought to specifically affect its DNA
polymerase activity, but possibly leave the remainder of
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Current Biology Vol 10 No 19
the protein undisturbed [1]. This mutant protein indeed
showed strongly reduced polymerase activity when
analysed in vitro and caused a cohesion defect in cells
almost like a deletion of the entire gene. Therefore it
seems likely that it is indeed the polymerase activity of
polymerase κ that is required for establishment and maintenance of sister chromatid cohesion.
What DNA does polymerase κ then replicate? This will be
an important question to address. One speculative model
is that the site where cohesin has pre-bound to DNA
might act as a replication fork barrier that can be traversed
only with the help of polymerase κ (Figure 1). This could
account for the requirement of polymerase κ for both chromosomal DNA replication and sister chromatid cohesion.
Or polymerase κ might be required to build specialised
DNA structures to which cohesin might bind? What are
the consequences of polymerase κ mutation on the
binding to chromosomes of the cohesin complex? Is continuous DNA synthesis by polymerase κ required to maintain an established chromosome cohesion site? There are
many questions that emerge from this groundbreaking
discovery about the requirement for polymerase κ. We can
look forward that their answers will bring us closer to an
understanding of how sister chromatids are tied together.
Acknowledgements
I thank S. Jones and J. Sgouros for sharing unpublished results, and A. Toth
and A. Verreault for helpful discussions on this dispatch.
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