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CELL CYCLE AND CELL DIVISION | 

Lead Editor: 
Zhaohua Tang, Ivor Hickey

Replication Fork Stalling and the Fork Protection Complex

By: Sarah A. Sabatinos, Ph.D. (Department of Molecular and Computational Biology , University of Southern
California) © 2010 Nature Education 
Citation: Sabatinos, S. A. (2010) Replication Fork Stalling and the Fork Protection
Complex. Nature Education 3(9):40

What happens at the DNA replication fork? How does a replication fork stall?
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DNA replication occurs during the synthesis (S) phase of the

cell cycle and starts at predefined DNA sequences known as replication origins;
this start is also known as origin firing (Errico &
Costanzo 2010; Koren et al. 2010). Once the origins of
replication have fired, the DNA replication proteins organize into a structure
called the replication fork (RF), where a group of proteins
coordinate DNA
replication (Langston et al. 2009). It
is called a fork because the simplified structure resembles a two-tined fork,
but its function is hardly simple, since it is the location of dynamic
activity
among a large group of proteins (Figure 1).

Figure 1: Replication fork components

The RF is a multiprotein complex with helicase and DNA

synthesis activities. It is called a fork because the structure
resembles a two-pronged fork. The helicase activities unwind
DNA in front of the fork to create regions of singled-stranded DNA
(ssDNA). The helicase components shown are the
minichromosome maintenance (MCM) helicase 2-7 hexamer,
CDC45, and associated GINS complex (simplified as a single
entity here). The ssDNA is coated in RPA (yellow circles) to keep
strands from reannealing. Fork protection complex (FPC)
components shown are Timeless (TIM), Tipin (TIPIN), Claspin
(CLASPIN), and And1 (AND1). Claspin (MRC1 in yeast) helps
connect the leading-strand polymerase epsilon (light blue circle)
to the helicase. And1 connects the lagging-strand polymerase
alpha (tan circle) to the helicase. Pol-alpha is part of the primase
complex, which synthesizes primers (thick tan line) on the lagging
strand. These primers allow the polymerase of the main lagging-
strand (polymerase delta, light green circle) to start synthesis.
The direction of DNA synthesis and RF movement into DNA is
indicated by arrows.
© 2010 Nature Education All rights reserved.

What Happens at the Replication Fork?

Two main activities happen at the fork: DNA unwinding and
DNA synthesis. The RF unwinds the unreplicated DNA ahead of it through a
helicase enzyme complex (Gambus et al.
2009; Lou et al.
2008). As their
name suggests, helicases modify the structure of the DNA helix and promote unwinding
and separation of the two DNA strands. The second activity of DNA synthesis at
the RF is
undertaken by DNA polymerase. This enzyme links together, or
polymerizes, DNA bases in the correct sequence using the template DNA strand, and
it generates two copies of the genome that are
later divided into daughter
cells in metaphase, or M phase (Langston et
al. 2009).

How Does a Replication Fork Stall?

A variety of impediments can stall the RF (Labib &
Hodgson 2007; Rothstein et al.
2000; Torres-Rosell et al. 2007). Functionally
this means that the RF complex is paused, sometimes indefinitely,
before
restarting. The cell's response to RF stalling depends on the number of stalled
forks and the length of the arrest (Labib & Hodgson 2007; Lucca et al. 2004; Meister et al. 2005; Tourriere &
Pasero
2007). By studying how RFs arrest and restart in the laboratory, we can learn how
defects in RF function can damage cells. Some chemicals can cause RF-stalling DNA
damage that leads
to diseases such as cancer (Bartkova et al. 2005; Calzada et al.,2005; Wray et al. 2008). Therefore scientists
study these drug effects on RF arrest in model organisms to understand the
effect on
DNA mutagenesis and cell viability.

Scientists study stalled RFs in the laboratory using the

drug hydroxyurea (HU). HU causes the depletion of deoxynucleotide triphosphates
(dNTPs) in cells (Bianchi et al.
1986; Koc et al. 2004;
Matsumoto et al. 1990). DNA polymerases use dNTPs
as building material for DNA synthesis and elongation. Without dNTPs, the
polymerase component of RFs cannot continue and slows down
and arrests (Feng et al. 2006; Mirkin & Mirkin 2007).

Because polymerase stalling causes RF complex arrest, these

RF activities are linked (Figure 2). In particular, the helicase-initiated
unwinding activity that precedes the RF is functionally linked to
the
polymerization activity (Calzada et al.
2005; Labib 2008; Lou et al. 2008;
Tanaka, Katou et al. 2009). To overcome RF stalling, the cell may adapt to or overcome
HU inhibition, or it may be
released from stalling when HU is removed from the
environment and the dNTP supply is replenished in the cell (Kurose et al. 2006; Lopes et al. 2001; Mulder et al.
2005). Irrespective of how long
the RF is arrested to make necessary repairs, RFs
must be capable of restarting so that the cell cycle can finish. To further
complicate matters, cells with stalled RFs are at an increased risk for
DNA
damage that can lead to mutation or cell death (Bernstein et al. 2009; Bryant et al. 2009; Froget et al. 2008; Kai et al. 2005; Mao et al. 2009; Noguchi et al. 2003; Petermann et al. 2010). Thus,
cells have distinct challenges in the response to and recovery from RF stalling
and can be studied using HU in laboratory experiments. How are stalled RFs
stabilized so that replication can
resume?

Figure 2: Keeping replication fork components together

Once origins have fired, the RF moves along DNA. The helicase complex unwinds DNA ahead of the fork, and the DNA
polymerases copy the DNA. In this figure, Errico and Costanzo (2010) demonstrate how the fork protection complex (FPC)
components (Tim, Tipin, And1, and Claspin) travel with the fork. If the FPC is not present during RF stalling, the helicase and
polymerase activities become separated, causing excessive ssDNA ahead of the RF and destabilizing the entire RF structure.
Courtesy of Dr. Alessia Errico. All rights reserved.

Setting the Stage: Considering Replication Fork Activity

When RFs stall upon dNTP depletion, how does the cell detect
this problem and respond? The RF helicase is a subcomplex of the fork complex
itself, and is made up of the hexameric
minichromosome maintenance (MCM)
helicase and several additional factors (see Figure 1; Gambus et al. 2009; Nedelcheva et al. 2005; Tanaka, Katou et al. 2009).
The MCM helicase travels with
the RF, unwinding DNA ahead of the RF to make single-stranded
DNA (ssDNA) available (Calzada et al.
2005; Langston et al. 2009;
Nedelcheva et al. 2005). ssDNA is
the substrate for both leading-
(Pole) and lagging-strand (Pold and Pola-primase)
polymerases. The ssDNA becomes coated with replication protein A (RPA), the ssDNA-binding
protein that ensures that the two separated DNA
strands remain separated.

The MCM unwinding activity must remain linked to

the polymerization activity to prevent excessive unwinding, which could also
cause DNA damage (Gambus et al.
2009; Nedelcheva et al.
2005;
Razidlo & Lahue 2008). If polymerization stops, the helicase has to stop
as well. A candidate complex to maintain this linkage is the fork protection complex
(FPC), which contains four
conserved proteins: Timeless, Tipin, Claspin and
And1 (Figure 2; Kemp et al. 2010;
Lou et al. 2008; Noguchi &
Noguchi 2007; Noguchi et al. 2004;
Unsal-Kacmaz et al. 2007;
Yoshizawa-
Sugata & Masai 2007). These fork stability components affect not
only proper RF function and the ability to stall, but also the ability of
chromosomes to segregate properly in metaphase

Timeless and Tipin Contribute to RF Replication and Stability

Timeless and Tipin (derived from the TIM and TIPIN genes) interact with other components of the RF and form the core of the FPC (Table 1 and Figure 2). These proteins have evolutionarily
conserved roles in DNA replication and RF stability (Errico et al. 2007; Gotter et al. 2007; Katou et al. 2003; McFarlane et al. 2010; Noguchi et al. 2003, 2004). In both yeast and humans, Timeless
and Tipin complex (also Tim/Tipin) promotes RF activity and is particularly important in DNA with repetitive sequences that are error prone (Dalgaard & Klar 2000; Krings & Bastia 2004; Noguchi &
Noguchi 2007; Razidlo & Lahue 2008). These naturally occurring DNA sequences that are at risk for pausing the RF even in the absence of external factors (such as HU) emphasize the importance
of RF stalling. Although not essential for DNA replication, Tim/Tipin promote promotes RF stability (Calzada et al. 2005; Errico et al. 2009; Gambus et al. 2006; Katou et al. 2003; Nedelcheva et al.
2005). Thus, these FPC proteins contribute to cell viability and mutational avoidance in undisturbed and deliberately stalled replication.

Table 1: Fork protection complex components and homologs from yeast to metazoa. The proteins of the
FPC are conserved from yeast to higher eukaryotes. Although the names are different between model
organisms, the function of each component within the FPC is similar. In addition, removal of a given
component often elicits the same phenotype; for example, loss of And1 (in frog, Xenopus laevis) or Ctf4 (in
yeast) affects chromosome cohesion.

Replication Protein Role in FPC References

fork homologs
 component In other
(human) species

Tim1 (Mouse,
Xenopus
laevis)
Fork stability; affects sister chromatid
Gotter et al., 2007; Leman et al., 2010;
TIM1 Tof1 (S. cohesion; maintains genome
McFarlane et al., 2010; Urtishak et al., 2009
cerevisiae) stability

Swi1 (S.
pombe)
Tipin (Mouse,
Xenopus
laevis)
Fork stability; has little effect on Errico et al., 2007; Gotter et al., 2007; Kemp
TIPIN Csm3 (S. DNA replication if removed; affects et al., 2010; Leman et al., 2010; Smith et
cerevisiae) sister chromatid cohesion al., 2009; Yoshizawa-Sugata & Masai, 2007

Swi3 (S.
pombe)
Claspin
Connects helicase and polymerase
(Mouse, Alcasabas et al., 2001; Calzada et al.,
epsilon; plays role in RF stability;
Xenopus 2005; Komata et al., 2009; Koren et al.,
involved in checkpoint signaling;
laevis) 2010; Lou et al., 2008; Naylor et al., 2009;
CLASPIN interacts directly with
Nedelcheva et al., 2005; Osborn & Elledge,
Mrc1 (S. CHK1/RAD53/CDS1 effector
2003; Szyjka et al., 2005; Tourriere et al.,
cerevisiae, kinases, as well as MUS81
2005; Zhao et al., 2003
S. pombe) nuclease

AND1
(Mouse,
Xenopus Connects helicase and polymerase
Bando et al., 2009; Errico et al., 2009;
laevis) alpha; has some effect on DNA
Gambus et al., 2009; Hanna et al., 2001; Im
replication, magnified with TIPIN
AND1 CTF4 (S. et al., 2009; Mamnun et al., 2006; Tanaka
depletion; loss affects chromatid
cerevisiae) et al., 2009; Williams & McIntosh, 2002;
cohesion and segregation; plays role
Zhu et al., 2007
MCL1 (S. in genome stability
pombe)

The FPC also assists in transmitting signals of RF stalling that help coordinate other steps in the cell cycle (Figure 3; McFarlane et al. 2010; Unsal-Kacmaz et al. 2007; Yoshizawa-Sugata & Masai
2007). For example, Tim/Tipin interacts with cohesin, the protein that holds sister chromatids together. Removal or disruption of Tim/Tipin disturbs sister chromatid cohesion, resulting in more
space between chromatids (Errico et al. 2009; Leman et al. 2010; Tanaka, Kubota et al. 2009). In these ways, Tim/Tipin influences DNA replication, fork stalling and the response to stalling, and
proper chromosome segregation in metaphase.

Figure 3: Intra-S phase checkpoint signaling

When replication stress is encountered, as during HU exposure, signals are transmitted through a kinase cascade. The paths
compared between species are shown, and the given proteins in the pathway have functional similarity between species. At the top,
signals are transmitted through the apical kinase: ATR in vertebrates; Mec1 in Saccharomyces cerevisiae (budding yeast); Rad3 in
Schizosaccharomyces pombe (fission yeast). These kinases form a complex with adaptor proteins such as Atrip (or Ddc2, or Rad26)
and transmit signals through transducers Claspin (or Mrc1 in yeast). For the purpose of this review, the ultimate target is the effector
kinase: CHK1 in vertebrates, Rad53 in budding yeast, and Cds1 in fission yeast.
© 2001 Nature Education Adapted from Alcasabas, A. A. et al. Mrc1 transduces signals of DNA replication stress to activate Rad53.
Nature Cell Biology 3, 958–965 (2001) 10.1038/ncb1101-958. All rights reserved.

Claspin Maintains Replication Fork Speed and Efficiency

Claspin is another component of the FPC that is involved in
multiple stages of DNA replication, particularly uninterrupted replication. We
know a great deal about its function from studying the
yeast strains that lack
the Claspin homolog Mrc1 (∆mrc1
mutants; Osborn & Elledge 2003). Interestingly, ∆mrc1 cells exhibit increased dormant origin firing (Koren et al. 2010), demonstrating the
role of
Mrc1 in regulating the start of replication. In addition, ∆mrc1 cells replicate DNA more slowly than wild type cells in
unstressed conditions (Szyjka et al.
2005), suggesting that Mrc1 function
is important for normal replication speed
and efficiency. Mrc1 associates with Tof1/Csm3 (Tim/Tipin homolog) linking the leading-strand
polymerase, Pole, to the helicase (Figure 2; Bando et al.
2009; Katou et al.
2003).

Mrc1 also conveys the ssDNA-RPA signal created by stalled RFs

to downstream signaling molecules and initiates the intra-S phase checkpoint (Tanaka
& Russell 2001, 2004; Zhao et al.
2003). For
example, Xu and colleagues found that fission yeast Mrc1 is
phosphorylated to recruit the effector kinase (Cds1) to the stalled RF, allowing
Cds1 to become active and stabilizing the stalled RF
(Figure 3; Xu et al. 2006). They demonstrated that Cds1
recruitment through Mrc1 (first step) is essential for Cds1 activation and
signal transmission (second step). This two-step model relies on
both the
presence of Mrc1 at the stalled RF and Mrc1 modification (phosphorylation) by
the upstream kinase Rad3, in response to RF stalling (Xu et al. 2006). This signal transduction via Mrc1,
and the
importance of Mrc1 at the RF, is similar in Saccharomyces
cerevisiae (Alcasabas et al.
2001; Branzei & Foiani 2006, 2007; Naylor et al. 2009; Osborn et al.
2002; Schleker et al. 2009;
Tourriere & Pasero 2007). Therefore Mrc1 is a replication-specific
mediator protein that brings together signaling components to transmit the
signal of RF stalling (Alcasabas et al.
2001; Osborn et
al. 2002).

However, Mrc1 modification/phosphorylation is also important

for its interactions with other RF components. Lou et al. (2008) discovered that Mrc1 phosphorylation changes its
interaction with
Polε at the RF. Hence, the Mrc1 and polymerase interaction is
flexible (Lou et al. 2008). During
RF stalling caused by HU, does Mrc1 phosphorylation alter the Mrc1-RF interaction
and allow
remodeling of other components at the RF?

The Role of And1

The last of the FPC proteins is And1, which links the lagging-strand
primase (Pola)
with helicase function (Figures 4 and 5; Gambus et al. 2009; Im et al.
2009; Miles & Formosa 1992; Williams &
McIntosh 2002). The yeast homologs
are Ctf4 and Mcl1 (Table 1; Miles & Formosa 1992; Williams & McIntosh
2002). And1 stabilizes Polα association with the RF, and it is important for normal
DNA replication (Figure 4; Errico et al.
2009; Tanaka, Katou et al. 2009; Tanaka, Kubota et al. 2009; Zhu et al. 2007). In yeast, the homolog Ctf4
links the helicase to Polα (Figure 5; Gambus et al.
2009).

This And1/Ctf4/Mcl1 trio also influences chromosome

segregation by promoting proper chromatid cohesion and centromere assembly
prior to metaphase (Hanna et al.
2001; Mamnun et al. 2006;
Tanaka,
Kubota et al. 2009; Williams & McIntosh 2002). And1 function in RF activity
and stability, coupled with its requirement to ensure proper chromosome
segregation, highlights the importance
of the FPC to later cell cycle events.

Figure 4: The role of And1 and Tipin at the replication fork

Errico and colleagues (2009) noted that Tipin and And1 are important components for RF stability. Tipin interacts with And1
and polymerase alpha (Pola) while AND1 binds to Tipin and helicase components and polymerase alpha. Thus, the And1 and
Tipin components of the FPC link polymerase alpha with the helicase. Errico et al. suggest that these FPC proteins are a
“flexible bridge” linking helicase and polymerase activities at the RF.
Courtesy of Dr. Alessia Errico. All rights reserved.
 Figure 5: The role of Ctf4 in promoting replication fork stability
The budding yeast And1 homolog, Cft4, links polymerase alpha to the RF, specifically the helicase (lower right, lagging strand).
Gambus et al. (2009) demonstrate in this paper that Ctf4 associates with helicase components (MCM proteins and also the
GINS complex). They also show that cells lacking both Ctf4 and Mrc1 (delta-ctf4 and delta-mrc1 mutants) cannot survive,
demonstrating how important the FPC is to cell health. If Mrc1 is not present (delta-mrc1), the Ctf4 component of the FPC
becomes critical to holding the RF together.
Courtesy of Dr. Agnieszka Gambus. All rights reserved.

Summary
HU-stalled RFs are stabilized by the presence of the FPC, which
includes Tim/Tipin, Clapsin, and And1 (Calzada et al. 2005; Noguchi et al.
2003; Tourriere & Pasero 2007). These proteins link
helicase and
polymerase activities, ensuring proper DNA replication and chromosome
segregation (Gambus et al. 2006; Leman
et al. 2010; Lopes et al. 2001; Szyjka et al. 2005; Tanaka, Kubota et
al.
2009; Urtishak et al. 2009;
Yoshizawa-Sugata & Masai 2009). The FPC provides a platform for damage response
signaling and mediates interactions with kinases that trigger an intra-S phase
arrest
(Boddy et al. 1998; Branzei &
Foiani 2007; Brondello et al. 1999;
Kumagai et al. 2004; Lee et al. 2005; Unsal-Kacmaz et al. 2007; Yoshizawa-Sugata &
Masai 2007). Research into the functions
of these proteins enables us to understand
how replication proceeds normally, and what mechanisms are used to stabilize
replication and allow successful restart and completion of S phase at a
later
time.

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BASIC INTERMEDIATE

Eukaryotes and Cell Cycle Aging and Cell Division

CDK Cell Cycle Control by Oncogenes and
Mitosis Tumor Suppressors: Driving the
Transformation of Normal Cells into
Cell Differentiation and Tissue
Cancerous Cells
Cell Division and Cancer
Collaboration of Mitotic Kinases in Cell
Cycle Control
ADVANCED
DNA Replication and Checkpoint Control
Coordination of Cell Cycle Events by Ran in S Phase
GTPase
Germ Cells and Epigenetics
Cytokinesis Mechanisms in Yeast
p53 : The Most Frequently Altered Gene in
Maternal mRNA and the PolyA Tail in Human Cancers
Oocytes
Regulation of mRNA Splicing by Signal
Recovering a Stalled Replication Fork Transduction
Replication Fork Stalling and the Fork Repairing Double-Strand DNA Breaks
Protection Complex
The DNA Replication Checkpoint and
Preserving Genomic Integrity During DNA
Synthesis
The Domino and Clock Models of Cell
Cycle Regulation
The Formation of Heterochromatin and
RNA interference
Turning Somatic Cells into Pluripotent
Stem Cells