DNA REPLICATION

UNIVERSAL FEATURES
OF DNA
1. Semiconservative
• Proved by Meselson and Stahl (1958) using heavy
(H) and light (L) isotopes of N
2. Specific Base
Pairing
3. Requirement for Primers
• For initiation of replication
4. 5’→3’ Direction of
Replication
• With respect to
the daughter
DNA strand
• 3’-OH as site of
attachment
of the next
nucleotide
5. Semidiscontinuous
• Leading and Lagging strands
6. Origin of
Replication
• Rich in AT pairs
• Replication
proceeds
bidirectionally
from the origin
7. Replisomes
• Collection of
proteins and
enzymes
involved in the
complex process
of replication
Acronyms: Naming Genes and Proteins
• Bacterial genes: 3 lowercase italicized letters →
function
• dna - gene involved in DNA replication
• uvr - … resistance to UV radiation
• rec - … recombination
• For genes that affect the same process,
use A, B, C...
• eg. uvrA, uvrB
• Gene products: not italicized.
• eg. dnaA (gene) → Dna A
(protein)
REPLICATION
PROTEINS:
Initiation Protein
• E. coli : dnaA proteins
(~20-30 dnaA)
• yeast: ORC protein
• functions:
• recognizes and bind the
origin of replication
(oriC)
• melting at origin
DNA Helicases (DNA unwinding
proteins)
• requires ATP to denature DNA helix
• binds ssDNA (not dsDNA)
• Two helicases in E. coli
• Helicase II
• attaches to template for lagging strand
• moves 5' → 3'
• Rep protein
• attaches to template for leading strand
• moves 3' → 5'
DNA Helicases (DNA unwinding
proteins)
• DNA helicase moves along one DNA strand only
SSBPs (Single-Strand Binding Proteins )
• Helix destabilizing proteins
• bind to sites where template has been unwound but
not yet duplicated
• functions:
• prevent reannealing and tangling of ssDNA
• prevent formation of 2° structures
• protect ssDNA from nuclease degradation
• In eukaryotes
• major SSBP is RPA (replication protein A)
SSBPs (Single-Strand Binding Proteins )
• “Ironing-out” of unwound DNA
Primase
s• Generates the RNA
primers
• Short chain
polyribonucleotide
(4-12 nt)
• Provides the
3’ –OH group
needed in DNA
chain elongation
DNA Polymerases
• Monomeric or heteromultimeric
• enzymatic activities:
• 5'→3' polymerization activity
• Elongation of daughter DNA strand
• processivity – property of DNA pol to remain
attached to the template and incorporate
nucleotides before detaching
• fidelity – 1 mistake per 109 or 1010 ntds
added
DNA Polymerases
• enzymatic activities:
• 3'→5' exonuclease activity
• proofreading
• 5'→3' exonuclease activity
• removes RNA primers at the 5' end
• not present in eukaryotic DNA polymerase
Bacterial DNA Polymerases
• In E. coli, there are five DNA polymerases:

Enzyme Gene Function

I polA Major repair enzyme
II polB Minor repair enzyme
III polC Replicase
IV dinB SOS repair and mutagenesis
V umuD’2C SOS repair and mutagenesis
Bacterial DNA Pol III
holoenzyme
• Replicates both strands simultaneously
• An aggregate of 10 proteins
• core enzyme:
• α (5’→3’ polymerization)
• ε (3’→5’ exonuclease
/proofreading)
• θ stimulates ε
• sliding clamp:
β2
• clamp loader:
(δδ’χψ)2τ2γ2
Bacterial DNA Pol III holoenzyme
Bacterial DNA Pol III
holoenzyme
• E. coli sliding clamp
• keeps DNA polymerase
on to allow duplication
of long stretches of
DNA
• Clamp loader
• Uses ATP to assemble
the sliding clamp to
the DNA
Assembling the Sliding Clamp
• Processivity is
achieved by the
sliding clamp
E. coli replication
fork…
DNA pol I

• 5’→3’ exonuclease activity can be removed by

treating DNA pol I with the protease subtilisin
• Small fragment: 5’→3’ exonuclease activity
• Large fragment: Klenow fragment
• With retained polymerase and 3’→5’
exonuclease activity
Klenow fragment
• Can be used in in vitro synthesis of ds DNA from
ssDNA
Eukaryotic DNA Polymerases
E. coli Eukaryotic Function
I Major repair enzyme
II Minor repair enzyme
β DNA repair
γ Mitochondrial DNA synthesis
III ε Leading strand synthesis
DnaG α Primase
δ Lagging strand synthesis
Eukaryotic Replication Fork
Eukaryotic PCNA
• Proliferating Cell Nuclear Antigen
• Analogous to the E. coli sliding clamp
DNA
Ligase
• Seals nicks using ATP or NAD
Topoisomerases
• Relaxes DNA;
removes DNA
supercoils
• In E. coli,
topoisomerase II or
gyrase is involved
in releasing the
final products of
circular DNA
replication
(decatenation)
STAGES OF
The Cell Cycle

Campbell, 2009
Stages of Replication
I. Initiation
• Recognition of position/s on the DNA molecule
where replication will begin
II. Elongation
• Synthesis of the complementary daughter
strand at the replication fork
III. Termination
• Completion of the process
Initiation: Origin of Replication
• Always begins at the same position/s in the DNA
molecule
• sequences rich in A-T pairs
• Prokaryotes/viruses – single origin (OriC)
• Eukaryotes – multiple origins
Initiation:
OriC
• ̴245 bp, made up of two parts:
• 13-nuc repeat motif (3x)
• consensus seq: 5'
GATCTNTTNTTTT 3'
• 9-nuc repeat motif (5x)
• consensus seq: 5ʹ TT A T A CA A A 3ʹ
T
C
C
Initiation:
OriC
Initiation: Replicons
• The genome is divided into replicons
• Segment of DNA that replicates from a single
origin of replication
Initiation: Replicons
• Prokaryotic replication
• Single origin, single replicon

source: Color Atlas of Genetics

Initiation: Replicons
• Eukaryotic replication
• Multiple origins, multiple replicons

source: Color Atlas of Genetics

Initiation: Steps

DnaA binds to 9-nuc repeat of OriC

torsional stress on DNA

melting of helix at the 13-nuc repeat of

OriC
Initiation: Steps

DnaBC attaches to melted

region;
DnaC later detaches

PRE-PRIMING COMPLEX

DnaB unwinds more DNA;

DNA topoisomerase releases
tension.
Initiation: SSBP attaches to
ssDNA
Steps
DnaG (a primase) attaches to
DNA

PRIMOSOME

Primase synthesizes RNA primer

DNA pol III dimer binds

(binding enhanced by sliding
clamp)

REPLISOME
Elongation and Termination
DNA pol III complements leading
and lagging strand simultaneously.

When DNA pol III arrives to

copy the template, RMPs detach
SSBP.

DNA pol I proofreads and replaces

primers.

DNA ligase seals nicks.

Termination of replication
MODELS OF
Replication Fork: Old Model
Replication Fork: New Model in
3D
• The DNA on the lagging strand is folded
• brings the lagging-strand DNA pol into a complex
with the leading-strand DNA pol
Replication Fork: New Model in
3D
• Lagging strand loops
to make both
polymerases move
in the same
direction
Replication Fork: New Model in
3D
• New insight on the trombone model (2013)
Termination
• defined termination sequence (ter element)
• 23bp consensus sequence
• binding site of ter protein which has contra-
helicase activity
• two growing points collide and termination
occurs
Replication Models for Circular
DNA
Model DNA
Theta Bacterial DNA
λ DNA
Rolling Circle F factor
ΦX174
DNA λ DNA
Replication Models for Circular
DNA
• Theta Model
Replication Models for Circular
DNA
• Theta Model
Replication Models for Circular
DNA
• Rolling Circle Model
Replication Models for Circular
DNA
• Rolling Circle Model
EUKARYOTIC
Origin of Replication: Eukaryotes
• For higher eukaryotes: ???
• For yeasts: ARS – autonomously replicating sequence
ARS Subdomains Function
A and B1 = ORS • binding site of ORC
B2 • melting
• binding site of ABF
B3
 torsional stress on the DNA
• ORS – origin recognition sequence
• ORC – origin recognition complex
• ABF – ARS binding factor
Origin of Replication: Eukaryotes
• Yeasts ARS
Primer Synthesis in Eukaryotes
• Eukaryotic primase and DNA pol α
• Tightly bound
• Cooperate in the synthesis of the first few
nucleotides for the new strand
• primase synthesizes an RNA primer (8-12 ntds
of RNA) then...
• DNA pol α extends the RNA primer by adding
~20 ntds of DNA then...
• elongation proceeds by DNA pol δ
Primer Synthesis in Eukaryotes
Elongation in Eukaryotes
• Ten eukaryotic DNA polymerases
• DNA pol α for primer synthesis
• DNA pol γ for mitochondrial
DNA replication
• DNA pol δ and ε work with PCNA (Proliferating
Cell Nuclear Antigen)
• Analogous to the prokaryotic sliding clamp
• FEN1 (flap endonuclease)
• removal of RNA primers
• no eukaryotic DNA pol with 5' → 3' exonuclease
activity
Accuracy of Replication
1. Specificity of base pairing
2. Pre-synthetic error control
• before the nucleotide is covalently added to the
growing chain, DNA pol
• undergoes a conformational change
• double-checks the exact base pair geometry
Accuracy of Replication
Accuracy of Replication
3. Proof-reading ability of DNA polymerase
• 3’5' exonuclease activity
• DNA repair mechanisms to correct replication
errors

• Error:
• 1 for every 108 to 1010 nucleotides
incorporated
 The End Replication
•Problem
For a linear DNA
molecules
• DNA replication
machinery is unable to
replicate the ends
• DNA molecules get
shorter, with potentially
disastrous
consequences for the
cell.
The End Replication
Problem
Telomeres
• Found at the terminal
regions of the
chromosomes
• Repeating unit:
TTAGGG
• Some functions:
• Protects genes from being eroded through
successive rounds of DNA replication
• Prevents fusion of separate chromosomes
 Telomere
•Replication
Telomerase
• Contains a telomerase RNA that serves as template
for telomere elongation
Telomere Replication