eukaryote

replication initiation

Difference from bacteria:

• Large genome
• chromosomes
• Thousands of replicons
with 10 folds difference in
length.
• Individual replicons are
presumably activated at
characteristic times
during S phase.

Figure 10.10: A eukaryotic chromosome

contains multiple origins of replication
that ultimately merge during replication.
Replicon firing must be coordinated = origins are selected for
initiation at different time points:
• If all fired at the same time, replication can be finished in an
hour. In reality, it takes more than 6 hours – only 15% are
replicated at the same time.
• Saccharomyces cerevisiae: default behavior is to initiate
early but cis-acting sequences can delay replication
• In general, replicons near active genes are replicated earliest
and replicons in heterochromatin replicate last.
• Regional activation patterns suggest that replicons near one
another are activated at the same time.

3
Visualize replication forks:
Replication forks appears as 100-300 foci rather than evenly
distributed: not fired at the same time and are spatially clustered.
DNA New DNA

FIGURE 10.11 Replication forks are organized into foci in

nucleus. Cells were labeled with BrdU. The left panel was
 Origins in S. cerevisiae
- Autonomous Replicating Sequence
(ARS)
- ARS elements are AT rich with
domains (B3, B2, B1, A)
- A contains a consensus sequence and is http://glossomics.blogspot.com/2012/11/the-
orc-origin-replication-complex-is.html
essential. A + any two B is sufficient.
- Only selected ARSs are used in a given cell cycle.
- Origin recognition complex (ORC)- multi-subunit DNA binding
complex that recognizes the ARS. Highly conserved – found in all
eukaryotes.
- ORC contacts A and B1 throughout most of the cell cycle (vs
DnaA)
- ORC binds to B2 and B3 only when its time to replicate
 Are origins cis-acting elements?
• Yeast origins satisfy the definition of cis-acting elements:
has consensus sequences that are recognized by proteins
• The conservation of ORC suggests conserved mechanism:
origins in other organism (= consensus sequences that are
recognized by ORC) should be conserved.
• However, no homology in origins between species
suggests origins are identified by complex mechanisms
• Suggested reconciliation: environmental factors can
change the use of origins
When nucleotides are insufficient to initiate replication
quickly enough, ORC can fall off from primary origin
and reinitiate from vicinity sequence (no consensus).
 Licensing factors
• ORC binds to origins throughout cell cycle
• Licensing factors are proteins recruited by ORC to initiate
replication.
• Licensing factors are inactivated or degraded upon
replication initiation to prevent reinitiation.

• (in this sense, DnaA is both ORC and licensing factor:

recognizes and binds to origin, initiates replication and is
inhibited and degraded after initiation)
quiz
1. How do bacteria replicate faster than C+D=60min?
a) Making multiforked chromosome
b) Synthesize DNA faster than 50k bp/min
c) Shortening C and D time
d) Making mini-cells
2. Correct statement:
a) Cyclin-dependent kinases change expression levels each
cell cycle
b) p53 can lead to either cell survival or programed cell
death
c) Terminally differentiated cells are arrested in G1 phase
d) Constitutively active Ras is a tumor suppressor
e) Ras-GDP is the active form of Ras
3. False statement:
a) Each replicon has one origin
b) Origin can be recognized by specific proteins
c) Eukaryotes have multiple origins
d) All origins in eukaryotes are fired at the same time
e) Origin is a cis-acting element
 DNA, but otherwise appears morphologically norm
cells form when segregation is aberrant; like minic
chromosome, but because septum formation is no
unaltered. This phenotype is caused by par (partit
(named because they are defective in chromosom
4. Mutations in which type of genes will lead to the shown
phenotype?
a) Genes that regulate septum positioning
b) Genes required for chromosome separation
c) Genes involved in DNA replication
d) Genes required for septum formation
e) Mutation in DnaA

FIGURE 9.5 Top panel: Wild-type cells. Bottom p

cell division under nonpermissive temperatures ge
multinucleated filaments.
 Lecture 2.2
Replication II - DNA synthesis
 Dissecting stages of replication
• Conditional lethal mutants: temperature sensitive alleles (why?)
• Two types of replication mutants:
– Quick-stop mutants:
Replication stops immediately upon temperature shifts.
Mutations in components of replication apparatus (proteins
that execute DNA synthesis)
– Slow-stop mutants:
Current replication can be completed but the next round
cannot be started.
Mutations in proteins exclusively involved initiation.
• Observation --> conclusion: there’re two stages of replication
that are accomplished by two sets of proteins.
• If there are proteins that act in both stages, which type of
mutants? Why?
 Two types of DNA synthesis
a. Synthesis involved in b. Synthesis involved in
genome replication DNA repair

OH-3

3 OH

Different polymerases are responsible for different types of

DNA synthesis
 DNA polymerases
• Polymerases for genome replication: Replicase
• Polymerases for repair

• Polymerases that function in large complex called

holoenzymes
• Polymerases that function independently

• Polymerases that have proof-reading activity

• Polymerases that are error prone
TABLE 11.1 Only one DNA polymerase is the replication enzyme.
The others participate in repairing damaged DNA, restarting stalled
replication forks, or bypassing damage in DNA.
E. Coli DNA polymerases
Enzyme Gene Function

I polA Major repair enzyme Most abundant

When replication is
II polB Replication restart
blocked by mutation
III polC Replicase

IV dinB Translesion replication Error prone

V umuD’2C Translesion replication Error prone

When researchers assay extracts of E. coli for their ability to

synthesize DNA, the predominant enzyme activity is DNA
polymerase I. Its activity is so great that it makes it impossible to
Eukaryote DNA polymerases
• Nuclear genome replication
• Organelle genome replication
• Priming (initiation) replication
• Repair
• Translesion replication
 DNA polymerases
One fundamental enzymatic activity shared by all polymerase:
Catalyzing the formation of phosphodiester bond between
matching base pairs. Direction: 5’ à 3’
Substrates:
1. nucleoside triphosphate/dNTP; provide energy for the reaction
2. 3’ –OH: requires a primer

3’
5’

3’
 DNA polymerase common structure
• Bacterial DNA polymerases have a large cleft composed
of three domains that resemble a hand.
• DNA lies across the “palm” in a groove created by the
“fingers” and “thumb.”
5’-3’
Palm: catalytic active site polymerization
Fingers: positions template domain
in the active site;
Thumb: binds DNA as it
exits enzyme = processivity
3’-5’
nuclease

20
 DNA polymerases: proof reading
Replicases often have a second
enzymatic activity:
• 3’ à 5’ exonuclease activity
• Structurally a different domain
from the synthesis domain.
• Substrate:
Mismatched base pair (lead to
structural changes in DNA)
 Types of replication errors

1. Substitution
• Effected by efficiency of proofreading
2. Frame shift
• Effected by processivity
• ability of an enzyme to perform multiple
catalytic cycles with a single template instead of
dissociating after each cycle (adding one dNTP).

22
 Fidelity of Replication

1. High-fidelity DNA polymerases

Replicases: geometry of free 3’-OH group is important
• precisely constrained active site that favors binding
of Watson–Crick base pair
2. Low-fidelity DNA polymerases
Repair polymerases
• Lower requirements on active site
• Can fit larger stretches of damaged or incorrectly
positioned nucleotides

23
 Two strands, two modes of synthesis

Okazaki
Fragments

24
 DNA Polymerase III holoenzyme
2 complexes/enzyme; 12 subunits (encoded by diff genes); 900 kD
Subcomplex components:
i. Catalytic core(2x):
- a subunit = DNA binding; Pol activity
(palm, fingers, thumb)
- e subunit = 3 -5 proofreading exo.
- q subunit = stimulates the exo.
ii. Dimerizing subunit:
t subunit = links the 2 cores
iii. Clamp(1x) - holds the Pol III catalytic core on DNA template
- each clamp is a homodimer of b subunits that
binds around DNA and ensures processivity;
- prevents Pol core from dissociating from template
iv. Clamp loader(1x) - g complex consisting of 5 proteins (c, y, d, d’, g)
• needed to place and release the clamp on DNA
• Energy consuming (ATP)
https://www.youtube.com/watch?v=0Ha9nppnwOc
26
 Replication steps
1. Separation of DNA strands: DNA Helicase (DnaB)
• Uses energy from ATP hydrolysis, 1ATP=melt 1bp
• typically hexamer with 6 binding sites for DNA
2. Single-strand DNA protection: Single-strand binding
protein (SSB)
• Binds ssDNA at replication fork; keeps them from
reforming duplex
• Binds as monomer, but the addition of more subunits is
cooperative
3. Primer synthesis: Primase (DnaG)
• Special RNA Pol (smaller than regular RNA Pol)
• Only synthesizes short stretches of RNA (11-12 nts)
4. DNA synthesis: DNA Pol III
5. Joining Okazaki fragments
a. Dipolymerase model- explains how leading and lagging
strands synthesized at same time
3’
5’ 1. Helicase creates rep fork (at ori)

5’
Lagging 3’
2

Leading
3’ 5’
Joining Okazaki fragments

1. Removal of RNA primer:

DNA Pol I (repair),
degrades RNA
fills gap with DNA

2. Nick ligation-DNA ligase

Okazaki Fragments joined by Ligase

• DNA ligase makes the bond that

connects the 3′ end of one
Okazaki fragment to the 5′
beginning of the next fragment.

Figure 11.22: DNA ligase seals

nicks between adjacent
nucleotides by employing an
enzyme-AMP intermediate.

30
https://www.youtube.com/watch?v=0Ha9nppnwOc
31
Role of ATP at the replication fork
i. for helicase (DnaB) to unwind duplex
ii. for Gyrase (a type of topoisomerase) needed
for relaxing, swiveling action
iii. for primase (DnaG) to start primer
iv. for DNA pol III: requires it for activation

32
Eukaryotic Genome Replication: DNA Polymerases

33
Eukaryotic Genome Replication: DNA Polymerases

More than 1 polymerase is active at any given time

Alpha (a)
-Primase activity
Delta (d)
-Replicates on lagging strand; has exonuclease
Epsilon (e)
-Replicates the leading strand; has exonuclease
34
Eukaryotic Genome Replication: What happens to the Histones?
Nucleosome & Replication
- Replication does not involve any protracted period of time in which
DNA is free of histones
- Replication fork displaces octomers
- Octomers dissociate into one H3-H4 tetramer; two H2A-H2B dimers

“old” H3-H4 tetramer is

randomly transferred to 1 of
the new DNA molecules
“old” or “new” H2A-H2B
are added to form the
octomer

35
Eukaryotic Genome Replication: What happens to the Histones?

- Histone modification persists through cell division

- The old histones serve as a template to guide the modification of
new histones; acetylation, methylation

Introduction to Genetic Analysis, 11th

Eukaryotic Genome Replication at the terminal Telomere
Telomeres
• Ends of linear eukaryotic chromosomes
• Facilitate pairing of homologous chromosomes
• Maintain integrity of chromosome:
Chromosomes w/o telomeres resemble dsDNA
breaks and repair proteins will attempt to ligate
two chromosomes together
• Progressively shorten as we age:
No primer for the very 5’ end

Genes XI
 Telomere protection
• A long series of short, tandemly repeated sequence,
100-1000x.
consists of a long series of short, tandemly repeated sequences. T-Loop
There can be 100 to 1,000 repeats, depending on the organism.
• 3’ end: G-T rich single strand
• Telomeric
T loopsequences
structure:can be written in the general form 5′-
(T/A)nGm-3′ where n is 1 to 4 and m is >1. FIGURE 7.28 shows a
Single
generic strand
example. 3’ end
One unusual loops
property of the back
telomeric sequence

to displace upstream
is the extension of the G-T–rich strand, which for 14 to 16 bases is
usually a single strand. The G-tail is probably generated because
complementary
there helix of the C-A–rich strand.
is a specific limited degradation

FIGURE 7.28 A typical telomere has a simple repeating structure

with a G-T–rich strand that extends beyond the C-A–rich strand.
The G-tail is generated by a limited degradation of the C-A–rich
strand.
 Telomere shortening
- a consequence of DNA
replication

- The lagging strand has

multiple primers; when the
last primer is removed
nucleotides are missing at
that end of the strand
creating a 3' overhang

- 3' overhang may be

snipped off and strand
becomes progressively
shorter

Introduction to Genetic Analysis, 11th

 Telomere maintenance
- Telomere shortening is prevented
by the action of telomerase

- Telomerase is a large
ribonuceoprotein = RNA + proteins
- RNA is complementary to the
telomerase G-rich sequence
- Protein is a reverse transcriptase;
DNA from a RNA template
• Telomerase isn’t perfect and in somatic (body) cells telomeres
shorten as cells age
• Telomerase is more accurate in stem cells and germ cells
• Cancer cells have a hyperactive telomerase; extremely long
telomeres