Eukaryotic DNA Replication

Figure 30.19 The eukaryotic cell cycle. The stages of mitosis and cell division define the M phase (M for
mitosis). G
1
(G for gap, not growth) is typically the longest part of the cell cycle; G
1
is characterized by rapid
growth and metabolic activity. Cells that are quiescent, that is, not growing and dividing (such as neurons), are said
to be in G
0
. The S phase is the time of DNA synthesis. S is followed by G
2
, a relatively short period of growth when
the cell prepares for cell division. Cell cycle times vary from less than 24 hours (rapidly dividing cells such as the
epithelial cells lining the mouth and gut) to hundreds of days. catenated

connected in a series of links, as in a chain

The mechanism of DNA replication in eukaryotic cells shows strong parallels with
prokaryotic DNA replication, but the situation in eukaryotes is vastly more complex.
For example, in a growing human cell, some 6 billion bp of DNA must be duplicated
with high fidelity once (and only once) each cell cycle. The events associated with
cell growth and division in eukaryotic cells fall into a general sequence having four
distinct phases, M, G
1
, S, and G
2
(Figure 30.19). Eukaryotic cells have solved the
problem of replicating their enormous genomes in the few hours allotted to the S
phase by initiating DNA replication at multiple origins of replication distributed along
each chromosome. Depending on the organism and cell type, replication origins, also
called replicators, occur every 3 to 300 kbp (for example, an average human
chromosome has several hundred replication origins). In lower eukaryotes such as
yeast, replicators are small discrete chromosomal regions (100-200 bp), but in
mammalian chromosomes, the zones where initiation of DNA replication occurs may
span 500 to 50,000 bp. So that eukaryotic DNA replication can proceed concomitantly
throughout the genome, each eukaryotic chromosome contains many units of
replication, so-called replicons.
Figure 30.20 Model for initiation of the DNA
replication cycle in eukaryotes. ORC is present at the
replicators throughout the cell cycle. The pre-
replication complex (pre-RC) is assembled through the
sequential addition of Cdc6p and MCM (a replication
licensing factor) during a window of opportunity
defined by the state of cyclin-CDK and Cdc7p-Dbf4p.
After initiation, a post-RC state is established. (Adapted
from Figure 2 in Stillman, B., 1996. Cell cycle control
of DNA replication. Science 274:165-1663)
Cell Cycle Control of DNA Replication
Initiation of replication involves
replicators and the origin recognition
complex, or ORC, a heteromeric protein
that binds to replicators. ORC is bound to
the replicators throughout the cell cycle.
Early in G
1
(just after M), ORC serves as
a landing pad for proteins essential to replication control. Proteins binding to ORC
establish a pre-replication complex (pre-RC), but the pre-RC can be formed only
during a window of opportunity during G
1
. One of the principal proteins in assembly
of the pre-RC in yeast is Cdc6p (the replication activator protein encoded by the
yeast cdc6 gene) (Figure 30.20). Once Cdc6p binds to ORC, replication licensing
factors (RLFs) that license or permit DNA replication to occur then bind to the
chromosomes. The RLFs render the chromosomes competent for DNA replication.
Two RLFs are required: RLF-B and RLF-M. It has been suggested that RLF-B is
confined to the cytosol and has access to the chromosomes only when the nuclear
envelope disappears early in mitosis. Apparently, RLF-B associates with ORC on the
chromosomes and is present at the beginning of G
1
. RLF-M is a heteromeric complex
of the MCM proteins (the name comes from mini-chromosome maintenance
because these proteins are essential in the maintenance of plasmids [mini-
chromosomes] in yeast cells). These various protein-protein interactions establish the
pre-RC, which consists of ORC, Cdc6p, the MCM complex, and other proteins.
At this point, two protein kinases act upon the pre-RC to directly trigger DNA
replication. One of these protein kinases is a complex of cyclin-dependent protein
kinase (CDK) and cyclin B, called cyclin B-CDK. (Cyclins are proteins synthesized
at one phase of the cell cycle and degraded at another; thus, the concentration of
cyclins cycles during the cell cycle. B-Cyclins accumulate at high levels just before S
phase.) Cyclin B-CDK can phosphorylate sites in ORC, Cdc6p, and several MCM
subunits. Phosphorylation of Cdc6p causes it to dissociate from ORC, whereupon it is
degraded. Some of the MCM also dissociates. Cyclin B-CDK also phosphorylates
Cdc7p-Dbf4p, the other protein kinase essential to activation of DNA replication.
Cdc7p-Dbf4p is a complex of a protein kinase encoded by the yeast cdc7 gene
(Cdc7p) and the product of the yeast dbf4 gene (Dbf4p) (Figure 30.20). One model
suggests that Cdc7p interacts with ORC and Dbf4p interacts with the replicator;
together, Cdc7p-Dbf4p phosphorylates the MCM complex. The consequence of these
actions brings the cell into S phase.
These phosphorylation events serve as a replication switch because once
proteins in the pre-RC are phosphorylated (and perhaps destroyed, as Cdc6p is), the
post-RC state is achieved. The post-RC state is incapable of re-initiating DNA
replication. This transformation ensures that eukaryotic DNA replication occurs once,
and only once, per cell cycle.
Eukaryotic DNA Polymerases
Figure 30.21 Structure of the PCNA
homotrimer. Note that the trimeric PCNA ring
of eukaryotes is remarkably similar to its
prokaryotic counterpart, the dimeric |
2
sliding
clamp (Figure 30.13). (a) Ribbon representation
of the PCNA trimer with an axial view of a B-
form DNA duplex in its center. (b) Molecular surface of the PCNA trimer with each monomer colored differently.
The red spiral represents the sugar-phosphate backbone of a strand of B-form DNA. (Adapted from Figure 3 in
Krishna, T. S., et al., 1994. Crystal structure of the eukaryotic DNA polymerase processivity factor PCNA. Cell
79:1233-1243. Photos courtesy of John Kuriyan, Rockefeller University )
A number of different DNA polymerases have been described in animal cells and
assigned Greek letters in the order of their discovery (Table 30.4). DNA polymerase
o, a complex multimeric protein, functions in the initiation of nuclear DNA
replication. Given a template, it not only synthesizes an RNA primer, but it then adds
deoxynucleotides to extend the chain in the 5' 3' direction. It consists of four
subunits: a 180-kD DNA polymerase, two associated primase subunits (60 kD and 50
kD), and another 70-kD subunit of uncertain function. DNA polymerase a lacks 3'
5' exonuclease activity. The processivity of DNA polymerase a is a modest 200
nucleotides or so. DNA polymerase o is the principal DNA polymerase in
eukaryotic DNA replication. It consists of a 125-kD catalytic subunit and a 50-kD
subunit that interacts with PCNA protein (for proliferating cell nuclear antigen). In
association with PCNA, DNA polymerase d carries out highly processive DNA
synthesis. PCNA is the eukaryotic counterpart of the E. coli |
2
sliding clamp; it
clamps DNA polymerase d to the DNA. Like |
2
, PCNA encircles the double helix,
but, in contrast to the prokaryotic |
2
sliding clamp, PCNA is a homotrimer of 37-kD
subunits (Figure 30.21). DNA polymerase d has 3' 5' exonuclease activity. DNA
polymerase c also plays a major role in DNA replication; its precise role is unclear,
but it may sometimes substitute for DNA polymerase o in lagging strand synthesis.
DNA polymerase | functions in DNA repair; DNA polymerase is the DNA-
replicating enzyme of mitochondria.
Table 30.4
The Biochemical Properties of Eukaryotic DNA Polymerases

o o c |
Mass (kD)
Native >250 170 256 3638 160300
Catalytic core 165180 125 215 3638 125
Other subunits 70, 50, 60 48 55 None 35, 47
Location Nucleus Nucleus Nucleus Nucleus Mitochondria
Associated functions
3' 5' exonuclease No Yes Yes No Yes
Primase Yes No No No No
Properties
Processivity Low High High Low High
Fidelity High High High Low High
Replication Yes Yes Yes No Yes
Repair No ? Yes Yes No
Source: Adapted from Kornberg, A., and Baker, T. A., 1992. DNA Replication, 2nd ed. New York: W. H. Freeman
and Co.
Reactions at the Eukaryotic DNA Replication Fork
Studies on animal viruses such as SV40 (SV for simian virus) have provided a useful
model for eukaryotic DNA replication. The SV40 genome (a 5-kbp, closed circular
DNA duplex with a single origin of replication) can be considered to represent a
single replicon. The virus makes extensive use of the host cells replication
machinery; only a single virus replication protein, T antigen, is viral-encoded. The
SV40 origin of replication is a 64-bp sequence consisting of at least three functionally
distinct domains: (a) a central set of four copies of the pentameric sequence GAGGC
organized as an inverted repeat. This sequence is the T antigen binding site. On one
side of the T antigen binding domain is (b) a 17-bp region composed exclusively of A
and T residues; on the other side is (c) a 15-bp imperfect inverted repeat. One T
antigen molecule binds to each of the four pentameric GAGGC repeats. The T antigen
is an ATP-dependent helicase, and once it forms a complex with the four pentameric
repeats, it enters the DNA duplex at the adjacent AT region and unwinds the helix.
The AT region, with only two H bonds per base pair, is susceptible to strand
separation. Helix unwinding creates two replication forks. Unwinding is facilitated by
replication protein A (RPA), a ssDNA-binding host cell protein that is the
eukaryotic counterpart of SSB. Viral DNA replication is bidirectional, and two
nascent chains are synthesized at each replication fork: a leading strand and a lagging
strand.
Figure 30.22 Model for
eukaryotic DNA
replication illustrating
polymerase switching and
Okazaki fragment
processing on the lagging
strand. (a) Leading strand
synthesis is initiated by
primase-DNA polymerase
o, and then RFC mediates
polymerase switching
(removal of DNA
polymerase a, assembly of
PCNA, and addition of
DNA polymerase o)not
shown. DNA helicase
unwinds the DNA at the
replication fork. On the
lagging strand, DNA polymerase a initiates synthesis through formation of an RNA primer (shown in red), followed
by a short deoxynucleotide oligomer. Then, RFC loads PCNA and a second DNA polymerase (o or c) and synthesis
of the Okazaki fragment continues. (b) When the DNA polymerase approaches the RNA primer of the downstream
Okazaki fragment, RNase H1 degrades this RNA by removing all but one of its ribonucleotides; FEN1/RTH1
removes this last one, and DNA ligase joins the two Okazaki fragments. (Adapted from Figure 1 in Bambara, R. A.,
Murante, R. S., and Henricksen, L. A., 1997. Enzymes and reactions at the eukaryotic replication fork. Journal of
Biological Chemistry 272:4647-4650)

Eukaryotic DNA Replication: Leading Strand Synthesis
Synthesis of the leading strand is initiated upon RNA primer synthesis by the primase
subunits of DNA polymerase o; then DNA polymerase a adds a stretch of DNA to the
primer (Figure 30.22). At this point, replication factor C (RFC), the eukaryotic
counterpart of the prokaryotic complex, carries out a process called polymerase
switching: RFC removes DNA polymerase o and assembles PCNA in the region of
the primer strand terminus. Then, DNA polymerase o binds to PCNA and carries out
highly processive leading strand synthesis.
Eukaryotic DNA Replication: Lagging Strand Synthesis
Lagging strand synthesis of Okazaki fragments is initiated in essentially the same way
as leading strand synthesis: synthesis is primed by DNA polymerase o, a
deoxynucleotide stretch is added by DNA polymerase o, and then switching to DNA
polymerase o takes place. Priming is a frequent event in lagging strand synthesis, with
RNA primers placed every 50 or so nucleotides. About 10-nucleotide lengths of RNA
primer are extended through addition of 10 to 20 deoxynucleotides by DNA
polymerase a before DNA polymerase o (or c) enters (Figure 30.22). All but one of
the ribonucleotides in the RNA primer are removed by RNase H1; then the
exonuclease activity of the FEN1/RTH1 complex removes the one remaining
ribonucleotide, and DNA ligase joins the Okazaki fragment to the growing DNA
strand.
T Antigen Function Is Regulated by Protein Phosphorylation
Phosphorylation at a single residue (Thr
124
) activates T antigen to bind at the SV40 ori
and initiate replication. This phosphorylation is catalyzed by a cyclin-dependent
protein kinase, CDC2-PK. Such kinases are implicated in triggering eukaryotic cells
to enter mitosis. Dephosphorylation at different amino acid phosphorylation sites on T
antigen also activates it. RPC, a protein involved in the early stages of SV40
replication, is a protein phosphatase subunit. The combined actions of CDC2-PK and
RPC generate an appropriately phosphorylated form of T antigen competent in
replication initiation. These various phosphorylations/dephosphorylations represent
paradigms for the central role of protein phosphorylation in regulating eukaryotic
DNA replication and cell division.
30.6 Replicating the Ends of ChromosomesTelomeres
and Telomerase
Figure 30.23 (a) In replication of the
lagging strand, short RNA primers are
added (pink) and extended by DNA
polymerase. When the RNA primer at
the 5'-end of each strand is removed,
there is no nucleotide sequence to read
in the next round of DNA replication.
The result is a gap (primer gap) at the
5'-end of each strand (only one end of
a chromosome is shown in this
figure). (b) Asterisks indicate
sequences at the 3'-end that cannot be
copied by conventional DNA
replication. Synthesis of telomeric
DNA by telomerase extends the 5'-
ends of DNA strands, allowing the
strands to be copied by normal DNA replication.
Telomeres are short (5-8 bp), tandemly repeated, G-rich nucleotide sequences that
form protective caps 1-12 kbp long on the ends of chromosomes (see Chapter 12).
Vertebrate telomeres have a TTAGGG consensus sequence. Telomeres are necessary
for chromosome maintenance and stability. DNA polymerases cannot replicate the
extreme 5'-ends of chromosomes because these enzymes require a template and a
primer and replicate only in the 5' 3' direction. Thus, lagging strand synthesis at the
3'-ends of chromosomes is primed by RNA primase to form Okazaki fragments, but
these RNA primers are subsequently removed, resulting in gaps (primer gap
Figure 30.23) in the progeny 5'-terminal strands at each end of the chromosome.
Telomerase, an RNA-dependent DNA polymerase (126 kD) whose
catalytic subunit showssubstantial homology to other reverse
transcriptases (see
Figure 30.24 The structure of AZT (3'-azido-2',3'-dideoxythymidine). This nucleoside is a
major drug in the treatment of AIDS. AZT is phosphorylated in vivo to give AZTTP (AZT 5'-
triphosphate), a substrate analog that binds to HIV reverse transcriptase. HIV reverse
transcriptase incorporates AZTTP into growing DNA chains in place of dTTP. Incorporated
AZTMP blocks further chain elongation because its 3'-azido group cannot form a phosphodiester
bond with an incoming nucleotide. Host cell DNA polymerases have little affinity for AZTTP.
Chapter 13 and below), maintains telomere length by restoring telomeres at the 3'-
ends of chromosomes. Telomerase is a ribonucleoprotein, and its RNA component
contains a 9- to 30-nucleotide-long region that serves as a template for the synthesis of
telomeric repeats at DNA ends. The human telomerase RNA component is 450
nucleotides long; its template sequence is CUAACCCUAAC. Telomerase uses the 3'-
end of the DNA as a primer and adds successive TTAGGG repeats to it, employing its
RNA as template over and over again (Figure 30.24, see also figure in Chapter 12
Human Biochemistry box: Telomeres and Tumors).
Telomeres A Timely End to Chromosomes? Mammalian cells in culture undergo
only 50 or so cell divisions and then they die. Somatic cells are known to lack
telomerase activity and, thus, they inevitably lose bits of their telomeres with each
cell division. Telomerase activity is missing because the telomerase-reverse
transcriptase gene (the TRT gene) is switched off. This fact has led to a telomere
theory of cell aging, which suggests that cells senesce and die when their telomeres
are gone. In support of this notion, a team of biologists headed by Calvin B. Harley at
Geron Corporation used recombinant DNA techniques to express the catalytic
subunit of human telomerase in skin cells in culture and observed that such cells
divide 40 times more after cells lacking this treatment have become senescent.
These results, though controversial, may have relevance to the aging process.