MB 4.4.

Replication of DNA in Eukaryotes

• Compaction of DNA in a eukaryotic
chromosome: Two
Linker DNA chromatids
of nucleosome (10 coils each)
Histone core
of nucleosome
One coil
(30 rosettes)

One rosette Nuclear

(6 loops) scaffold
One loop
(~75,000 bp)

30 nm Fiber

“Beads-on a-string”form of
chromatin

30 nm fiber, a higher-order DNA

organization of nucleosomes
• Nucleosomes are made up of histones, highly conserved basic proteins
specific to eukaryotes.
• Chromatin assembly factor (CAF-1) brings histones to the newly
synthesized DNA. The nucleosomes bound to the parental strand are
randomly dispersed among the daughter strands.

•The eukaryotic sliding clamp is

called proliferating cell nuclear
antigen (PCNA) is the site of CAF-1
binding.
Cont’d

Eukaryotic genome: multiple linear chromosomes consist of multiple

regions,each with its own origin (multiple replicons on each chromosome).

 Replicon is any piece of DNA which replicates as a single unit. It contains

an origin and sometimes a terminus.

A typical mammalian cell has 50000-100000 replicons with a size range of

40-200 kb. When replication forks from adjacent replication bubbles meet,
they fuse to form the completely replicated DNA. No distinct termini are
required.
Eukaryotic DNA Replication-Experimental systems

1. Small animal viruses (simian virus 40, 5 kb) are good mammalian models
for elongation (replication fork) but not for initiation.

2. Yeast (Saccharomyces cerevisiae): 1.4 X 107 bp in 16 chromosomes, 400

replicons, much simpler than mammalian system and can serve as a
model system

3. Cell-free extract prepared from Xenopus (frog) eggs containing high

concentration of replication proteins and can support in vitro replication.
Mechanism of DNA Replication in Mammals
The Replication is semi-conservative

 RNA primer is required

 Multiple origin of Replication in eukaryotic cells

 There are five classes of DNA Polymerases:

•DNA Pol α - Priming of DNA replication

• DNA Pol δ - Elongation of both strands
• DNA Pol β – DNA Repair
• DNA Pol ε - DNA Repair
• DNA Pol γ – Replication of Mitochondrial DNA
 - DNA Pol α is responsible for initiation of new strands.
- Elongation of a short piece of DNA,3-4 bp long, (the initiator DNA or
“iDNA”)

• A protein, Replication Factor C( RFC)- binds to iDNA and load DNA

Pol δ with its sliding clamp
(PCNA)
•  Proliferating cell nuclear antigen- a ring surrounding the DNA
• Human cell ,has 10,000– 100,000 different replication origins.
Initiation of DNA Replication

Clusters of about 20-50 replicons initiate simultaneously at

defined times throughout S-phase

 Origin of replication is bound by an ORC- protein

complex

 “ Licensing Factors” proteins bind to ORC and form

Protein-DNA complex (pre-replication complex)

 Helicase unwinds the DNA at the replication fork

Mammalian replication fork
Licensing factor:
• required for initiation and inactivated after use

• Can only enter into nucleus when the nuclear envelope

dissolves at mitosis

• Only initiate once per cell cycle

Licensing factor
Multiple origins of replication in Eukaryotes
Origin of replication in Yeast
-Yeast replication origins (ARS- autonomously replicating sequences,
enables the prokaryotic plasmids to replicate in yeast).

- Minimal sequence of ARS: 11 bp [A/T]TTTAT[A/G]TTT[A/T] (TATA box)

Additional copies of the above sequence is required for optimal efficiency.

- ORC (origin recognition complex) binds to ARS, upon activation by

CDKs (Cyclin Dependant Kinases) , ORC will open the DNA for
replication.
Unwinding DNA from parental nucleosomes before
replication : 50 bp/sec, helicases
New nucleosomes are assembled to DNA from a mixture of old
and newly synthesized histones after the fork passes.
Elongation: three different DNA polymerases are involved.
• DNA pol α : contains primase activity and synthesizes RNA primers for the
leading strands and each lagging strand fragments. Continues elongation
with DNA but is replaced by the other two polymerases quickly.

• DNA pol δ : on the leading strand that replaces DNA pol α. can synthesize
long DNA

• DNA pol ε : on the lagging strand that replaces DNA pol α. synthesized
Okazaki fragments are very short (135 bp in SV40), reflecting the amount
of DNA unwound from each nucleosome
Telomerase Replication

1)Contains a short RNA molecule as telomeric DNA synthesis template.

The ends of chromosomes contain 100-1000 repeating (TTAGGG)n
segments

2)Telomerase activity is repressed in the somatic cells of multicellular

organism, resulting in a gradual shortening of the chromosomes
with each cell generation, and ultimately cell death (related to cell
aging)

3)The unlimited prolieferative capacity of many cancer cells is associated

with high telomerase activity.
Other cells- Germ cells
-Immortalized cultured cells
 Rate of DNA synthesis and the need for multiple origins

Genome Fork S phase Origins Comment

speed

E. coli 4.6 Mbp 30 kb/min 40 min 1 S longer than

doubling time

Yeast 14 Mbp 7Kb/min 20 min 330 S would last 80hr if

(1 cm) only 1 ori

Human 3 Gbp 3 kb/min 7h >10 000 ? S would last 1 year

(2 m) if 1 ori
4.5. Regulation of DNA Synthesis

• DNA polymerase control the fidelity of DNA replication

• Proofreading refers to any mechanism for correcting errors

in protein or nucleic acid synthesis that involves scrutiny
of individual units after they have been added to the chain

• Processive DNA polymerases have 3’5’ exonuclease

activity
Home work

1) DNA Repair Mechanisms

2) Mutation
- Type of Mutation
- Causes of Mutation