*

*Eukaryotic DNA replication is not as well

understood as bacterial replication

*The two processes do have extensive similarities,

* Methylating Enzymes similar to the bacterial ones have also been
found in eukaryotes

*Nevertheless, DNA replication in eukaryotes is more

complex
* Large linear chromosomes
* Tight packaging within nucleosomes
* More complicated cell cycle regulation
Textbook of Biochemistry with Clinical Correlations, 7e edited by Thomas M. Devlin © 2011 John Wiley & Sons, Inc.
 *
n Eukaryotes have long linear chromosomes
n They therefore require multiple origins of replication
n To ensure that the DNA can be replicated in a reasonable time

n In 1968, Huberman and Riggs provided evidence

for the multiple origins of replication

n DNA replication proceeds bidirectionally from many

origins of replication
 (a) There are multiple origins of replication (ori) tandemly arrayed along each
eukaryotic chromosome. In humans they are spaced at approximately 50,000- to
100,000-bp intervals.
Figure 4.15 Tandem replicons and biodirectional replication in eukaryotes.
 *
n The origins of replication found in eukaryotes have
some similarities to those of bacteria
n Origin recognition complex (ORC)
n A six-subunit complex that acts as the initiator of
eukaryotic DNA replication
n It appears to be found in all eukaryotes

n Requires ATP to bind origins

n Single-stranded DNA stimulates ORC to hydrolyze ATP

 (a) The origin recognition complex (ORC,
yellow balls) binds to an origin. (b) A
minichromosome maintenance (MCM, red
balls) protein complex binds to this, cell
division cycle 6 protein (cdc6) is involved.
(c) The initiator complex is activated and the
helicase activity (green oval) opens the
parental strands to form a very small bubble.
SSB (green balls) binds to the single strands,
helicases enlarges bubbles.
(d) Polα/primase synthesizes the first RNA
primer and, after about 10 nt, switches to
elongating it with deoxyribonucleotides.
(e) After about 15–30 deoxyribonucleotides are
added, polα/primase leaves and the chain is
elongated by DNA polymerase δ
(f) An RNA primer is synthesized on the
discontinuous side of this replication fork, as in
normal fork progression.
(g) This primer is elongated as previously
described. Result: two replication forks diverging
from the origin. The process is symmetrical
around the axis indicated by the dotted line,
with mirror-image forks diverging from origin.

Figure 4.16 Initiation of replication at a eukaryotic replication origin.

 *
n Mammalian cells contain well over a dozen different
DNA polymerases

n Four: alpha (a), delta (d), epsilon (e) and gamma (g)
have the primary function of replicating DNA
n a, d and e à Nuclear DNA
n g à Mitochondrial DNA
n DNA pol a is the only polymerase to associate with
primase
n The DNA pol a/primase complex synthesizes a short
RNA-DNA hybrid
n 10 RNA nucleotides followed by 20 to 30 DNA nucleotides
n This is used by DNA pol d or e for the processive
elongation of the leading and lagging strands
n Current evidence suggests a greater role for DNA pol d

n The exchange of DNA pol a for d or e is called a

polymerase switch
n It occurs only after the RNA-DNA hybrid is made
 *

* The situation on the lagging strand is slightly more complicated in

eukaryotes than in E. coli (See Devlin Figure 4.14). A helicase activity
in eukaryotes separates the parental strands, and a single-strand DNA-
binding protein called replication protein A (RPA) binds to the exposed
single strands.
* In eukaryotes, the primase forms a complex with DNA polymerase α
(pol α) that initiates Okazaki fragment synthesis. This pol α/primase
complex synthesizes a primer of approximately 10 ribonucleotides,
and then switches from primase to DNA polymerase activity and
elongates the primer with approximately 15–30 deoxyribonucleotides.
* The product of this dual reaction is a short stretch of DNA covalently
attached to the RNA primer. Once the Okazaki fragment has reached
this length, the pol α/primase complex dissociates from DNA.
Replication Factor C (RFC) binds to this elongated primer and serves
as a clamp loader to assemble the PCNA sliding clamp.
* Then pol β binds to the PCNA and completes the Okazaki fragments to
a final length of about 130-200 bp.
*
* Primer removal is carried out in two steps by RNase H and
FEN1. Rnase H degrades the RNA primer, leaving a single
ribonucleotide attached to the end of the Okazaki fragment. Flap
endonuclease 1 (FEN1) removes the last ribonucleotide (and
possibly some deoxyribonucleotides) by peeling back one or a few
nucleotides to form a small “flap” and then cleaving the
phosphodiester bond at the angle to release the flap.
* If there is a mismatch within the first few nucleotides of the
Okazaki fragment, as a result of misincorporation by pol α, the
mismatch would destabilize the 5′-end and create a larger flap
that could be excised by FEN1. This increases accuracy of the
replication process by removing errors introduced by pol α.
* The gap that remains is filled by pol δ extending the 3-′end of
the more recently synthesized Okazaki fragment
Figure 4.14 Enzymes replicating the lagging strand in eukaryotes (leading strand not shown).
Textbook of Biochemistry with Clinical Correlations, 7e edited by Thomas M. Devlin © 2011 John Wiley & Sons, Inc.
n DNA polymerases also play a role in DNA
repair
n DNA pol b plays a role in base-excision repair
n Removal of incorrect bases from damaged DNA

n Recently, more DNA polymerases have been

identified
n Lesion-replicating polymerases
n Involved in the replication of damaged DNA
n They can synthesize a complementary strand over the
abnormal region
 *

n Replication doubles the amount of DNA

n Therefore the cell must synthesize more histones to
accommodate this increase

n Synthesis of histones occurs during the S phase

n Histones assemble into octamer structures
n They associate with the newly made DNA very near the
replication fork

n Thus following DNA replication, each daughter

strand has a mixture of “old” and “new”
histones
Newly made
histone proteins
 *

n Linear eukaryotic chromosomes have telomeres at

both ends

n The term telomere refers to the complex of

telomeric DNA sequences and bound proteins
n DNA polymerases possess two unusual features
n 1. They synthesize DNA only in the 5’ to 3’ direction
n 2. They cannot initiate DNA synthesis
n These two features pose a problem at the 3’ end
of linear chromosomes
 (a) The telomere replication problem. The 3’-end of one parental strand, the
template for the discontinuous (retrograde) daughter strand, is shown in dark tan,
the daughter strand is shown in light tan, and an RNA primer is in purple. The
problem is that there is no place to synthesize a primer that would allow the
daughter strand to be completed (region shown with????), so the daughter strand
will be shorter than the parental strand. Removal of the last RNA primer makes it
even shorter. (b) A telomere consists of many tandem repeats of a 6 nt sequence,
TTAGGG in humans, with the G-rich strand extending beyond the C-rich strand by
about 12–18 nt.

Figure 4.12 Human telomeres.

Textbook of Biochemistry with Clinical Correlations, 7e edited by Thomas M. Devlin © 2011 John Wiley & Sons, Inc.
n Telomeric sequences consist of
n Moderately repetitive tandem arrays
n 3’ overhang that is 12-16 nucleotides long

n Telomeric sequences typically consist of

n Several guanine nucleotides
n Often many thymine nucleotides
 Telomerase is a ribonucleoprotein complex with a short RNA strand as an
integral part; it catalyzes the addition of new 6-nt telomere repeats to the
3′end of a DNA chain. Telomerase RNA partially base-pairs with the
telomeric repeat and serves as the template for the reaction, while the
protein component functions as a reverse transcriptase, synthesizing DNA
using the RNA template. After a six-nucleotide repeat is added, the enzyme
can dissociate and bind again, and add additional 6-nt repeats.

Figure 4.17 Telomerase.

Textbook of Biochemistry with Clinical Correlations, 7e edited by Thomas M. Devlin © 2011 John Wiley & Sons, Inc.
 Step 1 = Binding

The binding-
polymerization- Step 2 = Polymerization
translocation cycle can
occurs many times

This greatly lengthens

one of the strands
Step 3 = Translocation

The complementary
strand is made by primase,
DNA polymerase and ligase

RNA primer
* http://web.centre.edu/bmb/movies/Telomere
s.html