DNA Replication

Lecture#4
By: Muhammad Matloob
Biomedical Scientist (NUST)
DNA Replication is Semi-
conservative
• No defined nucleus in prokaryotic cell so
replication occurs in cytoplasm.

• During replications, each strand of DNA

serves as template for the synthesis of new
strand
• Hypothesized by Watson and Crick
• Proven by Matthew Meselson and Franklin
Stahl (1957)
• Used N15 and N14 isotope of nitrogen to
identify newly synthesized strands
• [Link] cells were first grown in N14, then for a
short time moved to medium containing N15
DNA Replication in [Link]
• John Cairns used radioactively
labeled 3H for imaging of DNA in E.
coli cells
• He observed that DNA isolated
from cells during replication had
an extra loop
• Carins concluded that this was
due to DNA replication process
• The corners of this loop are
termed as replication fork
The Origin of Replication and DNA synthesis
• In experiments with λ-bacteriophage, it was revealed that DNA
replication starts at a unique points
• These points are rich in A=T sequences
• Thus are bound by double bonds
• These points in DNA are termed as origin of replication
• DNA synthesis always occurs from 5’ to 3’ end
• New bases are added to free 3’-OH group
• So how can both strand be synthesized simultaneously?
• This problem was solved by Reiji Okazaki et al. in 1960
Okazaki Fragments
• Okazaki concluded that both strands are synthesized simultaneously
• One strand is synthesized continuously (Leading Strand)
• Other stand is synthesized discontinuously (Lagging Strand)
• In Lagging strand, small pieces of newly synthesized DNA is formed
(Okazaki Fragments)
• In Prokaryotes 100-200 nucleotides
• In Eukaryotes 1000-2000 nucleotides
DNA is degraded by Nucleases and
Synthesized by Polymerases
• Nucleases or Dnases are enzymes that degrade DNA
• These enzymes are classified into two catagories
• Endonucleases: enzymes that can degrade DNA from a specific
internal site
• Restriction Endonucleases
• Cleave DNA at Specific internal sites
• Exonucleases: enzymes that degrade DNA from one end (5’3’ or
vice versa)
• E. coli contains at-least 5 different DNA polymerases
Replication process is very accurate
• It is necessary for survival of organism that nucleotide sequence in
replication should be with as few error as possible.
• Misleading can result in deleterious, perhaps lethal mutation.
• A mistake occurs only once in 109-1010 nucleotide
• This means one error per 100-1000 replications in E. coli
• Incorrect bases are rejected by DNA polymerase
• Incorrect nucleotide may be able to form H-bond, but does not fit in the
active site
• In reality, DNA polymerase adds incorrect nucleotide for every 104-105
correct ones
Proof Reading
• Error rate is reduced by additional enzymatic mechanisms
• Exonuclease activity of DNA Polymerase-I (Proofreading Activity)
• Translocation toward next nucleotide is inhibited
• Increases accuracy by 102-103 fold

• Polymerase III has two functions

• Proofreading activity(3’-5’ exonucleases) and polymerase activity (5’-3’).
• DNA polymerase check to make that bases on replication strand and newly
formed strand are complementary.
• If not then 3’-5’ exonucleases removes error in opposite to polymerization
Comparison of All DNA Polymerases in [Link]
Steps in Replication
COMPLEMENTARY STRAND SEPARATION
• In order for two complementary strand parental dsDNA to be replicated, they must
first separate (melt) on a small region..
• Because polymerase only act on ssDNA as a template.
• In prokaryotic cells replication begins at single, unique nucleotide sequence called
origin. ori in [Link].
• Its contains large AT-rich segments.
REPLICATION FORK FORMATION
• Replication is bidirectional (opposite direction) generating a replication bubble.
• Tines of fork represent the separated strand.
• REQUIRED PROTEINS
• A group of prepriming complex (a group of proteins) is required to start replication.
DNA Replication Requires Many Proteins
• 20 or more enzymes work together in DNA Replication
• Each with its own unique function
• The complex formed is termed as DNA replicase system or replisome
• Helicases: move along the DNA strand and unwind it using ATP
• Topoisomerases: relieve the stress created by Helicases on DNA
strands
• DNA-binding proteins: bind to ssDNA and prevent spontaneous
dsDNA formation
• Primases and Rnase-H1: formation and removal of RNA-primers
• DNA ligase: join nicks created by Rnase-H1 and Topoisomerases
DNA Polymerases
• All DNA polymerases have two requirements
1. Template
• Provide chemical basis of semiconservative DNA replication model
• First example of template guided biosynthetic reaction
2. Primer
• Complementary strand segment bound to ssDNA with free 3’-OH (primer
terminus)
• Mostly RNA
• DNA Polymerase have two sites
1. Insertion Site: for attachment of Free nucleotides
2. Post-Insertion Site: for attachment of nucleotide to DNA strand
Other DNA
Polymerases in [Link]
• DNA Polymerase-I: Discovered
first in [Link]
• DNA Polymerase-II: involved
primarily in DNA repair
• DNA Polymerase-III: principle
replication enzyme
• DNA Polymerase-IV and -V:
involved in unusual mechanisms
of DNA repair
Problem of Supercoils

• As the two strands are separated, a problem is encountered the

formation of
• Positive supercoils in region of DNA ahead of replication fork and
• Negative supercoils in the region behind replication fork.
• These accumulating positive supercoils interfere with further
unwinding of double helix.
• To solve this problem there is a group of enzymes called
topoisomerases.
• These are responsible for removing supercoils in helix by transiently
cleaving one or both of DNA strands.
Topoisomerase Type 1
• These enzymes reversibly cleave one strand
of helix
• They have both strand-cutting and strand-
resealing abilities.
• They don’t use ATP.
• They store energy from phosphodiester
bond cleavage and utilize in resealing.
• Each time a transient nick is created in one
strand and intact strand passes though it.
• Type I relax negative supercoils in [Link] and
both type of supercoils in other prokaryotes.
Topoisomerase Type 2
• These enzymes tightly bind to double helix and
cleaves both strands.
• And then enzyme makes a second stretch to pass
through it and reseals it.
• Both negative and positive supercoils can be
release through this ATP consuming process.
• DNA gyrase, a type II enzyme has an unusual
property of introducing negative coils so that
positive are neutralized by hydrolysis of ATP.
• This is helpful in replication during helix opening
and also in strand separation during
transcription.
DNA Replication Direction
• DNA polymerases are able to copy parental nucleotide sequences in 3’-5’
direction.
• Newly formed sequences are opposite 5’-3’ sequence in antiparallel strand.
• Both strand grow in 5’-3’ sequence one towards and one away from replication
fork.
• Mechanism is slightly different in both strand.
• Leading strand
• This strand is coping in the direction of replication fork.
• It is continuous.
• 2. Lagging strand
• This strand grows away from replication strand.
• It grows discontinuously, thus forming small okazaki fragments.
• These fragments eventually joins together by DNA ligase.
Replication Proceeds in Three Stages
• Initiation
• In E. coli, OriC is
• A=T rich
• Contains 5-repeats of specific 9-bp sequence
• At-least 10 different proteins/enzymes participate in this step
• Open helix of dsDNA
• Establish pre-priming complex
• This step is regulated such that replication occurs only once during
cell division
• DNA pols cannot initiate synthesis of complementary strand of DNA on a single
stranded template.
• Rather, require RNA primer, which is a short piece of DNA-RNA hybrid thus providing
a double stranded DNA-RNA hybrid.
• 3’ end of RNA serves as first acceptor of deoxynucleotide by action of a DNA pol.
[Link]
• A specific RNA polymerase called primase. synthesizes short stretches of RNA (10
nucleotides).
• These are complementary and anti parallel to DNA template.
• In resulting hybrid RNA-DNA duplex, uracil in RNA pairs with DNA.
• These short RNA sequences are formed constantly on lagging strand but one in
leading strand at origin.
• Substrate for this process is 5’-ribonucleoside triphosphates.
• Pyrophosphate is released as each ribonucleotide is added (as mono-ribonucleotide)
through formation of a 3’-5’-phosphodiester bond.
2. Primosome
• The addition of primase converts prepriming complex to a primosome.
Cont.
• Elongation
• Parent strand is unwound by Helicases
• Topological stress is removed by Topoisomerases
• ssDNA is stabilized through ssDNA-binding proteins
1. Leading Strand:
• Primase (Dna-G) adds RNA-primer
• DNA polymerase-III adds nucleotides to 3’-end with collaboration to Helicase
(Dna-B)
Cont.
2. Lagging Strand
• Accomplished by formation of short Okazaki fragments
• Primase synthesize a RNA-primer
• DNA-polymerase-III adds nucleotides to 3’-end
• Process is repeated for every Okazaki fragment
• Synthesis of Lagging strand is slower than that of Leading strand
• To cope with this, two DNA-polymerases act simultaneously on
Lagging strand, while one act on Leading strand
• DNA chain elongation is catalyzed by multisubunit enzyme, DNA poly III.
• DNA pol III begins to add nucleotides along single-stranded template in a
specific sequence in newly synthesized chain.
• DNA pol is highly processsive enzyme.
• This processivity is due to b-subunit of holoenzyme forming a ring that encircles
and move along template strand of DNA as a sliding clamp.
• Daughter cell is complementary and in 5’- 3’ sequence.
• Substrate for this process is 5’-deoxyribonucleoside triphosphates.
• Pyrophosphate is released as each deoxyribonucleotide is added to 3’-OH+
group of growing chain through formation of a 3’-5’-phosphodiester bond.
• Hydrolysis of PPi to 2Pi by phyrophosphatase means that a total 2 high energy
bonds are used to derive each deoxynucleotide.
• All four nucleotides ( {dATP}, {dTTP}, dGTP} and {dCTP} ) must be present in
chain to elongate if supply of anyone stops chain stops there.
• DNA polymerase continue to synthesize DNA on the lagging strand
until blocked by RNA primer.
• When this occur RNA is excised and gap is replaced by DNA
polymerase I
• 5’-3’ Exonuclease activity.
• Like polymerase III, monomeric poly I also have 5’-3’ exonucleases
activity that is able to hydrolytically remove the RNA primer.
• Exonucleases cut at end whereas endonucleases cleave internally.
• Firstly, DNA poly I locates the space (nick) between the 3’-end of
newly synthesized DNA and 5’-end of adjacent RNA primer.
• Next, DNA pol I hydrolytically removes the RNA nucleotides ahead of
.itself moving in the 5’-3’ direction(exonuclease activity)
• As it removes ribonucleotides, DNA pol I replaces them with
deoxyribonucleotides, synthesizing DNA.
• Then it proofreads and it continues until RNA primer is totally
degraded.
DNA LIGASES
• The final phosphodiester linkage between the 5’ phosphate group on
DNA synthesized by poly III and 3’-hydroxyl group on DNA pol I is
catalyzed by DNA ligase.
• The joining of these bond require energy which comes in most
organism through ATP.
Cont.
• Termination
• Two replication forks meet at terminus regions
• Contains specific sequence (multiple copies of 20-bp sequence) called
Tur
• This Tur sequence functions as binding site for Tus-proteins
• This Tur-Tus complex halts the replication process
• Prevents over-replication of DNA by one fork if other is delayed
DNA Replication in Eukaryotes
• Similar yet more complex than prokaryotes
• Essential features of DNA-replication are same
• Eukaryotic replication is well coordinated with cell-division
• Introduces more complexities
• Cyclins and Cyclin-dependent Kinases ensure that replication occurs
once during cell division
• These cyclins are degraded after mitosis
• Rate of movement of replication fork is 1/20th times compared to E.
coli
• Humans contain multiple Origin of replication to counter this
Cont.
• As in prokaryotes, eukaryotes contain multiple polymerases with
different functioning
• DNA polymerase ε synthesizes the leading strand
• DNA polymerase δ synthesizes the lagging strand
• Both enzymes have 3′→5′ proofreading exonuclease activities.
• DNA polymerase α synthesizes RNA primers
• Extends them by about 10 nucleotides of DNA
• No proofreading 3′→5′ exonuclease activity
• Replication termination is characterized by synthesis of telomers at
the end of chromosomes