CHAPTER 1
Conserved Steps in Eukaryotic
DNA Replication
XIN QUAN GE AND J. JULIAN BLOW
Wellcome Trust Centre for Gene Regulation and Expression, University of
Dundee, DD1 5EH, UK
1.1 Overview: the Biochemistry of DNA Synthesis
The genome of mammals comprises B6 109 nucleotides arranged in extremely long linear polymers—the chromosomes. Accurate copying of this
amount of genetic information in a biologically relevant time frame (often a few
hours, though sometimes as little as a few minutes) requires highly accurate
enzyme machines together with complex molecular coordination and feedback.
The DNA template is a long polymer of four types of deoxyribonucleotide
arranged as a double-stranded anti-parallel helix (Figure 1.1A). The backbone
of each strand consists of phosphodiester linkages between the 3 0 and 5 0 carbons of deoxyribose. The 1 0 carbon of the deoxyribose is linked to one of four
different bases: the purines (adenine and guanine) or the pyrimidines (thymidine and cytosine). The two strands are held together by hydrogen bonds
between complementary bases. The two-ringed heterocyclic purines always
base pair with single ring pyrimidines, maintaining the linear axis of the helix
and avoiding backbone distortion; specifically, guanine forms three hydrogen
(H) bonds with cytosine and adenine makes two hydrogen bonds with thymidine (Figure 1.1B). Whilst each H bond is relatively weak, the huge number of
H bonds in an average mammalian chromosome (4109) ensures that the duplex
is extremely stable. As noted by Watson and Crick in 1953,1 each of the two
Molecular Themes in DNA Replication
Edited by Lynne S. Cox
r Royal Society of Chemistry 2009
Published by the Royal Society of Chemistry, www.rsc.org
1
2
Figure 1.1
Chapter 1
The chemistry of DNA synthesis. (A) The duplex DNA template showing
the phosphodiester backbone, with the hydrophobic bases facing inwards,
forming complementary base pairs. Reproduced from ‘An overview of the
structure of DNA’, created by Michael Ströck 2006, released under the
GNU Free Documentation Licence (GFDL).a (B) The four nucleotides of
DNA form hydrogen bonds with their complement (note opposite polarities of the two strands). Adenine pairs with thymidine via two H bonds,
while guanine pairs with cytosine through three H bonds. (C) Phosphodiester bond formation through nucleophilic attack from the 3 0 OH of a
newly incorporated nucleotide onto the a phosphate of an incoming
nucleotide. The pyrophosphate released is rapidly hydrolysed by pyrophosphatase to inorganic phosphate, with a highly negative DG (B40
kJ mol1). a
single strands of DNA contains all the information necessary to produce new
second strands through complementary base pairing.
During DNA replication, the two strands are opened up and a nascent
strand, complementary to the template strand, is synthesised by a complex of
many proteins called the replication fork or replisome. Unwinding of the two
strands of DNA to expose bases during template-directed DNA synthesis
requires the input of chemical energy to break the hydrogen bonds. This energy
is derived from hydrolysis of ATP by helicases, which act as DNA-dependent
ATPases that run ahead of the replication fork (Figure 1.2). In eukaryotes, the
major replicative helicase is almost certainly the hexameric Mcm2-7 complex2–5
(Figure 1.3, see also Chapter 3). At the same time, torsional stress (positive
supercoiling) is caused by unwinding the DNA and this is relieved by topoisomerases (nicking-closing enzymes) (Figure 1.2). Then DNA is synthesised by
enzyme-mediated polymerisation of deoxyribonucleotide triphosphates
a
http://en.wikipedia.org/wiki/File:DNA_Overview.png
3
Conserved Steps in Eukaryotic DNA Replication
Topoisomerase
Helicase
Figure 1.2
DNA duplex being unwound during DNA replication. Double-stranded
DNA is being separated into two single strands by a helicase moving from
right to left. The topological interlinks between the two strands are
removed by a topoisomerase.
LEADING STRAND
3'
5'
Mcm2-7
helicase
DNA polymerase
5'
3'
RNA primer
Okazaki
fragment
LAGGING STRAND
Figure 1.3
3'
5'
Leading and lagging strand replication. A replication fork is shown moving
from right to left. The double-stranded template DNA is unwound by the
Mcm2-7 helicase. The leading strand is synthesised continuously in the 5 0 –
3 0 direction. The lagging strand is also synthesised in the 5 0 –3 0 direction
with respect to the nascent strand, but since this is opposite to the overall
direction of fork movement, it is synthesised discontinuously in Okazaki
fragments. Each Okazaki fragment is started by a small RNA primer
which is subsequently removed before the fragments are ligated together.
(dNTPs) complementary to the sequence of the exposed bases on the template
strand (see Chapter 4). New daughter duplexes thus consist of one parental
template strand base paired to a complementary daughter strand. This is semiconservative replication and was first demonstrated experimentally by Meselson and Stahl,6 who showed that newly synthesised DNA is composed of one
template strand plus one nascent strand.
During polymerisation, nucleophilic attack by the lone pair of electrons on
the 3 0 hydroxyl (OH) of deoxyribose onto the 5 0 phosphate of an incoming
4
Chapter 1
dNTP results in the formation of a phosphodiester bond with the elimination of
pyrophosphate (Figure 1.1C). The subsequent, and very rapid, hydrolysis of
pyrophosphate to two inorganic phosphates releases energy, and it is this that
drives the polymerisation reaction forwards. An important consequence of this
reaction is that DNA must always be synthesised in a 5 0 to 3 0 direction; all
known DNA polymerases act 5 0 –3 0 with respect to the newly synthesised
(nascent) DNA molecule. However, while the double-stranded DNA template
exists as an anti-parallel double helix, replication of both template strands is
coordinated at the replication fork, which moves in a net direction away from
the start point (replication origin). To overcome this conflict of directionality,
on only one strand (the ‘leading strand’) can DNA be polymerised in the same
direction as the fork is moving. On the other strand (the ‘lagging’ strand),
nascent DNA is synthesised in short sections called Okazaki fragments, typically B150 nucleotides long in eukaryotes (Figure 1.3). Okazaki fragments are
started by short RNA primers which are subsequently removed before the
fragments are joined together. Thus the fork can move away from the start site
while co-coordinating synthesis of both nascent strands and without contravening the energy requirements of 5 0 –3 0 synthesis.
In eukaryotes, the chromosomal DNA is located within the cell nucleus
where it is associated with proteins to form a DNA-protein complex called
chromatin (see Chapter 10). The basic building block of chromatin is the
nucleosome core particle, which contains 147 base pairs of double-stranded
DNA wrapped in 1.65 left-handed superhelical turns around the surface of
histone octamer comprising two central H3–H4 dimers flanked on either side
by two H2A–H2B dimers (Figure 1.4). A variety of other proteins also bind to
DNA and regulate its activity. For replication to occur, pre-existing nucleosomes and other DNA-bound proteins that are located ahead of replication
forks need to be transiently disrupted. After fork passage, those proteins are
deposited back on parental as well as nascent DNA so that the chromatin
status is reproduced in daughter strands7 (see also Chapter 10).
1.2 Where and When Does DNA Replication
Take Place?
1.2.1 Cell Cycle Control
In eukaryotes, DNA replication takes place during a distinct phase of the cell
cycle called S phase, during which time the entire genome is precisely duplicated
(Figure 1.5). The replicated DNA molecules are segregated to the two daughter
cells during a subsequent cell cycle phase called mitosis (see Chapter 9). S phase
and mitosis are separated by two ‘gap’ phases, G1 and G2. Progression through
each stage of the cell cycle is very tightly regulated by a complex interplay of
kinases (enzymes that phosphorylate proteins), phosphatases (enzymes that
remove phosphate groups from proteins) and proteases (which degrade proteins into shorter polypeptides or constituent amino acids).
5
Conserved Steps in Eukaryotic DNA Replication
A.
H3-H4 dimer
H2A-H2B dimer
B.
Figure 1.4
Structure of the nucleosome. (A) Cartoon of the nucleosome, showing 1.65
turns of DNA wrapped around an octamer consisting of two H2A-H2B
dimers and two H3-H4 dimers. (B) Crystal structure of the nucleosome
with DNA: DNA in turquoise and brown, core histones H3 (blue),
H4 (green), H2A (yellow) and H2B (red). Reprinted by permission
from Macmillan Publishers Ltd: K. Luger, A. W. Mader, R. K. Richmond,
D. F. Sargent and T. J. Richmond, Crystal structure of the nucleosome
core particle at 2.8Å resolution, Nature, 1997, 389, 251–260, copyright
(1997).84
During S phase, pairs of replication forks are initiated bidirectionally from
chromosomal loci called replication origins. The large size of eukaryotic
chromosomes (each of which can be tens or hundreds of megabases long) means
that in order for them to be replicated in a reasonable period of time, a large
number of replication origins are needed. Although the initiation of a pair replication forks at a replication origin is a tightly controlled process, each fork will
typically then move along the DNA (‘elongate’) until it encounters a fork moving
6
Chapter 1
Origin Licensing
(Mcm2-7 loading)
Timing
Decision
Point
anametaMitosis
G1
G2
S
Inhibition of
Licensing
Re
Figure 1.5
pli
cat
io
n of
Differe
al D
nt Chromosom
s
ain
om
DNA replication and the cell cycle (schematic view of events). In the metaphase of mitosis (M, meta-) condensed chromosomes (consisting of paired
chromatids) are aligned on the metaphase plate by the mitotic spindle.
During anaphase (M, ana-) the two sister chromatids are pulled into two
daughter cells. During late mitosis and G1, replication origins are licensed
by loading Mcm2-7 complexes. Origin licensing is inhibited at other cell
cycle stages. During early G1, specific regions of chromosomal DNA take
up specific positions in the nucleus, with open chromatin (red) tending to
be positioned internally, and more condensed chromatin (blue) tending to
be positioned at the nuclear periphery and in larger internal structures. At
this time (the timing decision point), these different chromosomal regions
become programmed to replicate at different stages of S phase (green).
in the opposite direction, at which stage both forks will disassemble (‘terminate’).
When DNA is visualised during the S phase, replicated DNA can be observed as a
series of ‘bubbles’ with replication origins near their centres (arrowheads in Figure
1.6). The stretch of DNA replicated by forks emanating from a single origin is
Conserved Steps in Eukaryotic DNA Replication
7
0.1µM
Figure 1.6
Replication bubbles. Electron microscopic image of replication origins in
developing Drosophila embryos. Replication bubbles are indicated by the
arrowheads. Scale bar 0.1 mM. Reprinted from: G. Micheli, C. T. Baldari,
M. T. Carri, G. Di Cello and M. Buongiorno-Nardelli, An electron
microscope study of chromosomal DNA replication in different eukaryotic systems, Experimental Cell Research, 137, 127–140, copyright (1982),
with permission from Elsevier.85
referred to as a replicon. Replicon sizes can vary significantly, both among different organisms and among different cell types in the same organism. Rapidly
dividing cells typically have small replicon sizes (for example, cells in the early
Xenopus embryo has an average replicon size of B10 kb, whilst mammalian
somatic cells typically have replicon sizes of 50–150 kb8–10).
1.2.2 Origin Clusters and Replication Foci
In metazoans, adjacent origins (typically 2–5) are organised into clusters which
initiate synchronously while different origins clusters are activated at different
stages of S phase.11 One or more clusters of origins are organised into a discrete
replication focal site, which has been estimated to comprise about 1 Mb of DNA
and 6–12 replicons. Each focus is thought of as a factory for DNA replication
and contains a range of replication fork proteins (forming so-called replisomes).12 It is possible that replisomes are anchored to a fibrous network within
the nucleus (the ‘nuclear matrix’ or ‘nuclear scaffold’) through which multiple
replication forks are spooled; alternatively, the physical organisation of the
chromosomal DNA into higher order chromatin structures could provide the
framework on which replication foci are built.13–15 DNA replication is typically
completed in each focus within 30–120 minutes,16 and during this time, live cell
imaging of the replication fork protein PCNAi (see Chapters 3 and 7) has shown
that replication foci do not merge, divide or have directional movement,17,18
thus arguing that replication foci are achieved by the coordinated assembly and
disassembly of replisomal proteins at sites that are more or less fixed.
1.2.3 The Replication Timing Programme
Eukaryotes replicate their genomic DNA according to a specific temporal
programme, with different clusters of origins firing at different time during an S
phase that lasts from minutes in yeast to hours in metazoans. Several pieces of
evidence have suggested that chromatin context is a critical determinant of
origin initiation time. The replication timing programme is re-established in
i
Proliferating cell nuclear antigen
8
Chapter 1
19
each cell cycle shortly after mitosis. Transcriptionally active regions tend to
have open chromatin structure and replicate early, whereas gene-poor regions
and the more condensed heterochromatin replicate late.20,21 Transcriptional
silencing can reprogramme an origin from initiating early to late, as well as by
promoting a more compact chromatin structure around the region.22
The timing decision point in early G1 (Figure 1.5) is the time when specific
regions of the chromosome become programmed to replicate at specific stages
of S phase. This takes places coincidently with the repositioning of chromosomes in the nucleus and the formation of immobile structures in the nucleus
that restrict chromosome mobility.19,23 It has been proposed that chromatin
regulators might be concentrated into subnuclear compartments by a clustering
of related chromosomal domains, which may influence the timing of origin
firing within a chromatin domain. For example in yeast, late replicating
origins reside close to the nuclear periphery in G1, whereas early replicating
origins are apparently randomly localised within the nucleus throughout the
cell cycle.24
Many other factors could also contribute to determining the timing of origin
firing. For example, in Saccharomyces cerevisiae, the timing of replication in
certain origins is shown to be affected by the origin sequence.25 Precise levels of
cyclin-dependent kinase (CDK) activity present at various stages of S phase are
important for executing the temporal programme. In budding yeast, two S
phase cyclins have differential roles in activation of early and late origins: Clb5
activates both early and late origins, while Clb6 activates only early origins.26
The replication timing programme determines the differential firing time of
large sequence blocks containing replication origin clusters, but why has the cell
evolved such a sophisticated programme for DNA replication? The grouping of
replication forks into factories that are activated at different times might provide an environment whereby newly replicated DNA could be assembled into
specific chromatin states, thus maintaining the epigenetic information that is
important for regulation of other nuclear activities (such as transcription).10,27
It may also allow for tight regulation feedback, for example blocking firing of
late origins when replication from early origins is halted.
1.3 Origins of DNA Replication
The number of origins ranges from a few hundred in a yeast cell to tens of
thousands in a human cell. The extent to which conserved DNA sequence
elements determine origins differs significantly among eukaryotic species.
Replication origins in the budding yeast Saccharomyces cerevisiae contain
highly conserved sequence elements called A, B1, B2 and B3 boxes of the
autonomously replicating sequence (ARS).28 These conserved DNA sequences
are required for binding of the initiator protein ORC (origin recognition
complex, see Chapter 2). However, not all DNA segments containing the
conserved sequence elements are recognised by ORC in vivo. Other sequences
distributed over 100 bp also contribute to replication origin function, possibly
Conserved Steps in Eukaryotic DNA Replication
9
by providing binding sites for proteins that can enhance the recruitment of
ORC to DNA or by providing DNA sequences that can be easily unwound.29
The origins in most other eukaryotes are much less stringent in terms of
sequence requirement. In the fission yeast, Schizosaccharomyces pombe, the
required origin sequences are distributed over large DNA segments (500–
1000 bp) and are AT rich.30 It appears that it is the number of AT tracts in a
given segment of DNA that determines its probability of binding ORC and
functioning as an origin of DNA replication.
The nature of origins in metazoans is even less well defined than in yeasts and the
origins appear not to contain any consensus sequence. Replication origins occur at
frequent and nearly random intervals along metazoan chromosomal DNA, and
only a fraction of them are utilised in each cell cycle with a wide variation of
efficiency. A typical pattern of origin initiation in metazoans is broad zones containing many relatively inefficient origins, one or a few of which are selected stochastically and the rest are suppressed.31,32 However, at some origins, such as
lamin B2 and b-globin origins, replication starts from tightly-defined sites.33,34
Several interacting components may influence the location and efficiency of
initiation in any given cell cycle, such as:
(1) DNA sequences. Sequences rich in AT could facilitate ORC binding or
DNA unwinding.
(2) Local chromatin structure. It has been shown that the positions of
nucleosomes near origins are important for origin function.35 Whilst
histone acetylation has been shown to affect origin specification in
Xenopus and Drosophila,36,37 in mammalian cells, an ATP-dependent
chromatin remodelling complex is required for efficient replication of
heterochromatin.38
(3) Transcription. Transcription has been shown to interfere with origin
activity and indeed, replication origins are almost never found within
actively transcribed DNA.10,20,39,40
(4) Protein–protein interactions. The presence of other proteins could help
recruit ORC and enhance origin efficiency. For example, Abf1 and the
Myb protein complex bind to origins and can affect the efficiency of
origin utilisation in yeast and Drosophila.41,42
(5) Origin interference. It has been observed that in an initiation zone, firing
of one replication origin appears to inhibit initiation at nearby origins,
but is coordinated with neighbouring origins at more distant sites.43 This
may suggest some sort of long range interaction between origins.
1.4 Licensing of DNA for Replication
It is essential for a cell to replicate its genome only once per cell cycle and this is
regulated by the ability of cells to load the Mcm2-7 protein complex onto the
origins (see Chapters 2 and 3). Mcm2-7 form a clamp around DNA and provide
helicase activity to separate the double helix ahead of replication forks2–5 (see
Chapter 3). During late M and G1 phases of the cell cycle, Mcm2-7 are loaded
10
Chapter 1
Replication
Licensing System
Active
M
M
M
M
M
Replication
Licensing System
Inactive
R
M
+ ge
M
L
G2
∆C
d t1
CDK
G1
S
m i nin
M
M
S
M
M
M
Free Mcm2-7
M
Mcm2-7
On DNA
Active
Mcm2-7
Helicases
M
Figure 1.7
The replication licensing cycle. The replication licensing system (RLS) is
activated in late mitosis, promoting the loading of Mcm2-7 double hexamers onto DNA. During S phase, DNA-bound Mcm2-7 becomes activated as helicases. When replication forks terminate, Mcm2-7 are released
from DNA. In metazoans, the licensing system is shut down in S phase
and G2 by degradation of Cdt1 and activation of the Cdt1 inhibitor,
geminin; in early mitosis, high CDK levels also inhibit licensing. In yeasts,
licensing inhibition in S, G2 and early mitosis is directly mediated by high
CDK levels.
onto the DNA, which probably involves the clamping of the proteins around
origin DNA without activation of their helicase activity (Figure 1.7). This
‘licenses’ the origin for use in the subsequent S phase. Mcm2-7 loading requires
the recognition of the origin DNA by the origin recognition complex (ORC)
(Figure 1.8). ORC in turn recruits proteins Cdc6 and Cdt1, which load Mcm2-7
onto DNA by hydrolysing ATP44 (see Chapter 2). The complex of ORC, Cdc6,
Cdt1 and Mcm2-7 at replication origins is termed the pre-replicative complex or
pre-RC. It is not clear whether ORC, Cdc6 and Cdt1 open the Mcm2-7 ring and
load it around DNA, or whether they facilitate the assembly of the Mcm2-7
hexamer on DNA from different Mcm subcomplexes present in the nucleoplasm.
As a licensed origin initiates during S phase, the Mcm2-7 complex becomes
activated as helicase, possibly by binding other replication fork proteins
including the GINS complex.45,46 Since Mcm2-7 proteins travel with the replication fork,47,48 this means that an origin becomes unlicensed after it initiates. To
prevent DNA being replicated a second time in a single cell cycle, it is therefore
important to prevent re-licensing of replicated origins during S and G2 phases of
the cell cycle. The mechanisms for achieving this vary in different eukaryotes.
In yeasts, CDKs which are active from late G1 to mid-mitosis, prevent
licensing outside late M and G1 phase by ORC inactivation, Cdc6/Cdt1
11
Conserved Steps in Eukaryotic DNA Replication
ORC
A
Cdc6
Cdt1
ORC
B
M
Cdc6
C
dt1
ORC
M
Figure 1.8
M
Origin licensing. Cartoon showing steps in the licensing of a replication
origin. (A) ORC association with DNA at the replication origin. (B) ORC
recruits Cdc6 and Cdt1. (C) ORC-Cdc6-Cdt1 allows the loading of multiple Mcm2-7 complexes onto the DNA.
degradation and Mcm2-7 export.49 In metazoans, the main route by which
licensing is prevented during S and G2 is the downregulation of Cdt1 activity.
This is brought about both by degradation of Cdt1 protein and activation of a
Cdt1 inhibitory protein, geminin. Cdt1 is degraded at the end of G1 and early S
phase in a process dependent on SCF-class ubiquitin ligase and cul-4 ubiquitin
ligase.50–53 When geminin builds up during S, G2 and M phase, Cdt1 is stabilised by binding to geminin. As a result, licensing is inhibited and Cdt1 is
protected from degradation, so that when geminin is degraded in late mitosis
and G1, Cdt1 is ready for licensing.49,54
ORC can load multiple copies of Mcm2-7 complexes onto DNA, and Mcm27 are inB20-fold excess over replication origins used in S phase.55–60 Cells
synthesise DNA at normal rates when the level of Mcm2-7 is reduced61,62 and,
in Xenopus egg extracts, normal replication rates are maintained when Mcm2-7
complex is reduced toBtwo per origin.59,60,63,64 This suggests that each of the
loaded Mcm2-7 complexes could act at an origin to initiate DNA replication
and up to 90% of them remain dormant in a single S phase. It has recently been
shown that a biological role of the excess Mcm2-7 complexes loaded during
licensing is to maintain genomic stability.65 When forks stall during DNA
replication, the dormant Mcm2-7 complexes can initiate and rescue DNA
replication between two stalled forks, allowing the intervening DNA to be
12
Chapter 1
replicated. If a replication fork encounters an unfired (dormant) origin, the
Mcm2-7 must be removed from it to prevent re-replication from occurring.
1.5 Initiation of DNA Replication
Mcm2-7 is loaded in an inactive form at replication origins during G1 (Figure
1.9A), and then activated to initiate DNA replication during S phase. Activation of Mcm2-7 requires both S phase CDK (cyclin E/A-Cdk2) and Dbf4dependent kinase (DDK) activity (Figure 1.9B). DDK and CDK are expressed
at relatively constant levels during the cell cycle, but the expression of regulatory subunits (Dbf4 and cyclin respectively) is increased in S phase. One of
the known substrates of Cdc7-Dbf4 is the Mcm2-7 complex, the phosphorylation of which is thought to change its interaction with other replication fork
proteins.66 Recently, it has been shown in budding yeast that S phase CDKs
phosphorylate two replication proteins, Sld2 and Sld346,67 (Figure 1.9B). By
contrast, the critical S phase substrates of CDK-cyclin activity in metazoans
have not yet been identified. Phosphorylation of yeast Sld2 induces its interaction with Dbp11, and facilitates its association with the GINS complex (Sld5,
Psf1, Psf2, Psf3) and Dbp11 (Figure 1.9C). At the same time, Sld3 is phosphorylated by CDK and recruited to DNA by binding to Cdc45, where
phospho-Sld3 recruits Dbp11 and Sld2. Current evidence suggests that the
binding of Cdc45 and GINS to Mcm2-7 activates the helicase activity of
Mcm2-7.45,46 Consequently GINS and Cdc45 remain associated with Mcm2-7
and travel with the active replication forks (see Chapter 3).
1.6 Elongation of Replication Forks
Mcm2-7 and associated complex unwind the DNA in a bidirectional manner
away from origins, and the single-stranded DNA (ssDNA) becomes coated with
a binding protein called Replication Protein A (RPA). Via interactions with the
helicase and RPA, DNA polymerase a (pola) is loaded onto the template
(Figure 1.9D). A subunit of the pola holoenzyme provides primase activity and
synthesises short RNA primers (8–12 nucleotides long), which are then extended
by the DNA polymerase activity of pola to synthesise a short initiator DNA
(iDNA) of about 30 bases. Because pola does not have a proofreading exonuclease activity, the iDNA synthesised only serves as a DNA primer for more
extensive DNA synthesis by DNA polymerases with proofreading activity after
polymerase switching (Chapter 4). The primer-template DNA structure is
recognised and bound by a clamp-loading heteropentameric protein complex,
replication factor C (RFC). This promotes structural changes in RFC, which
uses the energy from ATP hydrolysis to open the ring of the trimeric sliding
clamp PCNA (Chapter 2), and clamp it around the DNA, while at the same time
pola is displaced. PCNA acts as a processivity factor for the elongation DNA
polymerases pold and pole by forming a ring that tethers them to the template
DNA (Chapters 3, 6 and 7). Current data suggest that pole is on the leading
13
Conserved Steps in Eukaryotic DNA Replication
Licensed
Origin
A
3'
5'
5'
3'
Mcm2-7
CDK
B
P
P
Sld2
Sld3
Cdc7
P
Dpb11
P
Sld2
Sld3
P
3'
5'
5'
3'
P Dpb11 P
Sld3
Sld2
G
Cdc45 I N polε
P S
P
C
3'
5'
5'
3'
D
Figure 1.9
Cdc45
5'
3'
G
N polε
I
p o lδ p o lα
S
P
3'
5'
G
polα
polδ
p o lε I
S
N C dc45
Initiation of replication forks. A model for events occurring during S phase
as an origin initiates DNA replication. (A) A licensed origin, loaded with
two Mcm2-7 hexamers. (B), Cdc7 phosphorylates members of the Mcm2-7
complex, whilst CDKs phosphorylate Sld2 and Sld3. Phosphorylated Sld2
and Sld3 associate with Dpb11. (C) The Sld2-Sld3-Dpb11 complex
associates with phosphorylated Mcm2-7, possibly via interactions between
Sld3 and Cdc45 and between Sld2 and GINS/pole. (D) Mcm2-7 helicase
activity unwinds the origin DNA, allowing pola to initiate synthesis of the
two nascent strands. These nascent strands are elongated by pole to form
the leading strand of the fork. Behind this, pola and pold act together to
synthesise Okazaki fragments on the lagging strand.
14
Chapter 1
68,69
strand and pold is on the lagging strand.
When the lagging strand polymerase encounters the 5 0 end of the adjacent Okazaki fragment, the 5 0 end is
displaced to form a 5 0 flap, which is degraded by endonuclease FEN1 (Chapter
5). Then the two Okazaki fragments are ligated by DNA ligase and the DNA
polymerase is recycled to a newly loaded clamp on the lagging strand.70
Recent work in budding yeast indicates that Mcm2-7 associate with a
number of proteins at forks to form the ‘replisome progression complex’ (RPC)
during elongation,71 perhaps to ensure that fork progression is coordinated
with DNA synthesis and other processes. In addition to Cdc45 and GINS, the
RPC also contains Ctf4 which is important for establishing cohesion between
sister chromatids, Tof1-Csm3, which mediate pausing of forks at DNA replication fork barriers, the checkpoint mediator Mrc1, the histone chaperone
FACT, topoisomerase 1 and Mcm10.72 In addition, PCNA acts as a landing
pad for other proteins during replication such as the CDK inhibitor p21,
cytosine methyltransferase, the chromatin assembly factor CAF-1, DNA ligase,
FEN1 and other proteins involved in DNA repair73–75 (see Chapter 3).
1.7 Termination of DNA Replication
Replication forks terminate when they encounter another replication fork
coming from the opposite direction. In most cases, this occurs without the need
for any special DNA sequences. In some cases, though, replication fork barriers
at specific DNA sequences slow replication forks so that these sites are likely to
become sites of termination. One such example is in the heavily transcribed
ribosomal DNA genes, where the replication fork barrier is positioned to
inhibit replication forks from moving through the gene in the opposite direction
from transcription.76
The exact mechanism of how replication machinery is displaced from the
DNA during termination is poorly understood. At termination, the replication
forks must be disassembled. Most of the proteins released from terminated
replication forks can be recycled to newly initiating forks. The Mcm2-7 proteins, however, are a special case. They are released from DNA at termination,
but are not reloaded onto DNA until the next mitosis in order to prevent DNA
from being replicated more than once in a single cell cycle (Figures 1.5 and 1.7).
Similarly, if an active fork encounters inactive Mcm2-7 bound to a dormant
replication origin, the inactive Mcm2-7 will be displaced from DNA.
1.8 Replication of Chromatin
The histones around which the DNA is wrapped (Figure 1.4) have to be displaced from the chromatin as the DNA replication forks pass, and the newly
synthesised DNA has to be reassembled into chromatin (see Chapter 10).
Nucleosome disruption is likely to be facilitated by ATP-dependent chromatin
remodelling enzymes, such as WSTF which is targeted to replicating DNA
through direct interaction with PCNA and in turn recruits ISWI-type
Conserved Steps in Eukaryotic DNA Replication
15
77
nucleosome-remodelling factor SNF2. At the same time, histone chaperones
facilitate the disruption of parental nucleosomes by acting as histone acceptors
and hence aid the transfer of the histones onto the nascent strand.78 FACT is
complexed with Mcm proteins during fork movement, and facilitates nucleosome disruption and re-deposition of H2A-H2B.79,80 CAF-1 associated with
PCNA is aided by Asf1 to deposit H3-H4 onto replicating DNA.81,82
Chromatin also contains epigenetic information in addition to that from the
DNA sequence which affects, amongst other things, the level of gene expression. This information is encoded by covalent modifications to histones (such
as acetylation and methylation) as well as methylation of cytosine bases. When
DNA is replicated, the epigenetic information must be copied too. This is
achieved by association of a large number of chromatin-modulating enzymes
with PCNA during replication such as DNA methyltransferase I, CAF-1 and
histone deacetylase (Chapters 3 and 10). These enzymes either themselves have
catalytic activity or can recruit other enzymes implicated in chromatin
modification.
1.9 Chromatid Cohesion and Segregation
After replication, it is essential that the sister chromatids are identified and each
of them is sent to a different daughter cell. To achieve this, replicated chromosomes remain physically attached to each other by cohesion until anaphase,
when they are separated by microtubule pulling force. Sister chromatid cohesion is established by cohesin, a complex consisting of at least four proteins
(Smc1, Smc3, Scc1, Scc3) that form a ring structure loaded at discrete sites
along the entire length of the chromosome in G1 phase. During S phase, the
cohesin complex establishes a physical link (cohesion) between replicated sister
chromatids by several factors, including Eco1, Ctf4 and Ctf18. Several models
have been proposed to explain how cohesin contributes structurally to sister
chromatid cohesion, one of which is that the cohesin ring establishes cohesion
by embracing both sister chromatids (see Chapter 9).
At the metaphase-to-anaphase transition, the separation of sister chromatids
is triggered by the removal of cohesin from chromosomes. This is achieved by
activation of a protease called separase, which cleaves the cohesin ring.
Separase is inhibited by protein securin and, at the metaphase-to-anaphase
transition, securin is degraded after APC/C dependent ubiquitination.83 Thus a
complex interplay of cell cycle regulatory factors establishes cohesion concomitant with synthesis of sister chromatids and ensures separation only at the
metaphase-anaphase transition (Chapter 9).
Acknowledgements
The authors are supported by Cancer Research UK grants C303/A4416 (XQG)
and C303/A7399 (JJB).
16
Chapter 1
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