General DNA replication

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BIOLOGY 305 SECTION 3
LECTURE NOTES
ALLEN JOHNNY BORLAY
(BSc, MSc)
COURSE INSTRUCTOR
DNA REPLICATION
GENERAL OVERVEW
 In both prokaryotes and eukaryotes, DNA replication
occurs as a prelude to cell division.
 This DNA replication phase is called the S phase
(synthesis phase).
 The two daughter DNA molecules formed from
replication eventually become chromosomes in their
own right in the daughter cells.
 As with all phenomena that involve nucleic acids, the
basic machinery of DNA replication depends on
complementarity of DNA molecules and on the ability
of proteins to form specific interactions with DNA of
specific sequences
GENERAL OVERVEW
 Copying genetic information for transmission to
the next generation
 Process of DNA duplicating itself
 Process of duplication of the entire genome
prior to cell division
 Begins with the unwinding of the double helix to
expose the bases in each strand of DNA
 Each unpaired nucleotide will attract a
complementary nucleotide from the medium
– will form base pairing via hydrogen bonding
 Enzymes link the aligned nucleotides by
phosphodiester bonds to form a continuous
strand
Three alternative patterns for DNA replication
 In semiconservative replication,
each daughter duplex contains
one parental and one newly
synthesized strand.
 Dispersive replication results in
daughter duplexes that consist of
strands containing only segments of
parental DNA and newly DNA newly
synthesized DNA
 In conservative replication, one
daughter duplex consists of two
newly synthesized strands, and the
parent duplex is conserved
Meselson and Stahl Experiment
 Experiment allowed differentiation of parental and
newly formed DNA
 Bacteria were grown in media containing either
normal isotope of nitrogen (14N) or the heavy
isotope (15N)
 DNA banded after equilibrium density gradient
centrifugation at a position which matched the density of
the DNA
 When bacteria grown in 15N were transferred
to normal 14N containing medium,
– the newly synthesized DNA strand had the 14N
while the parental strand had 15N
Meselson and Stahl Experiment cont.
Meselson and Stahl Experiment cont.
• They checked the composition of
the resulting DNA molecules by
density gradient centrifugation
found an intermediate band
indicating a hybrid molecule
containing both 14N and 15N DNA.
PROKARYOTIC DNA REPLICATION
Replication in prokaryotes starts at a
single origin of replication
 The rate of replication in prokaryotes
is faster (10×) than in eukaryotes
 The number (3) of DNA polymerases is
less than that in eukaryotes
 Approximately 1000 nucleotides are
added per second (In E.coli)
 Addition of nucleotides requires energy
similar to ATP
PROKARYOTIC DNA REPLICATION
BASIC STEPS
 DNA unwinds at the origin of replication
 Helicase opens up the DNA-forming replication forks→ these are
extended bidirectionally
 Single-strand binding proteins coat the DNA around the
replication fork to prevent rewinding of the DNA.
 Topoisomerase binds at the region ahead of the replication fork
to prevent supercoiling.
 Primase synthesizes RNA primers complementary to the DNA
strand.
 DNA polymerase starts adding nucleotides to the 3'-OH end of
the primer.
 Elongation of both the lagging and the leading strand continues.
 RNA primers are removed by exonuclease activity.
 Gaps are filled by DNA pol adding dNTPs.
 The gap between the two DNA fragments is sealed by DNA ligase,
which helps in the formation of phosphodiester bonds.
ILLUSTRATION
Enzymes/Proteins and Their Function in Prokaryotic DNA Replication
Enzyme/protein
Specific Function
DNA pol I
Exonuclease activity removes RNA primer and replaces with
newly synthesized DNA
DNA pol II
Repair function
DNA pol III
Main enzyme that adds nucleotides in the 5'-3' direction
Helicase
Opens the DNA helix by breaking hydrogen bonds between the
nitrogenous bases
Ligase
Seals the gaps between the Okazaki fragments to create one
continuous DNA strand
Primase
Synthesizes RNA primers needed to start replication
Sliding Clamp
Helps to hold the DNA polymerase in place when nucleotides
are being added
Topoisomerase
Helps relieve the stress on DNA when unwinding by causing
breaks and then resealing the DNA
Single-strand binding
proteins (SSB)
Binds to single-stranded DNA to avoid DNA rewinding back.
POINTS TO . NOTE
 Leading strand →continuously synthesized strand
 The overall direction of the lagging strand will be 3' to 5', and
that
of the leading strand 5' to 3'.
 Lagging strand → strand that is replicated in short fragments
and away from the replication fork
 Okazaki fragment → DNA fragment that is synthesized in short
stretches on the lagging strand
 Replication fork → Y-shaped structure formed during initiation
of replication
 Primer →short stretch of nucleotides that is required to initiate
replication → in the case of replication → the primer has RNA
nucleotides
 Origins of replication → specific sequences of nucleotide where
replication
SUMMARY
 Replication in prokaryotes starts from a sequence found on the
chromosome called the origin of replication—the point at which the
DNA opens up
 Helicase opens up the DNA double helix, resulting in the formation of
the replication fork.
 Single-strand binding proteins bind to the single-stranded DNA near
the replication fork to keep the fork open.
 Primase synthesizes an RNA primer to initiate synthesis by DNA
polymerase, which can add nucleotides only in the 5' to 3' direction.
 One strand is synthesized continuously in the direction of the
replication fork; this is called the leading strand. The other strand is
synthesized in a direction away from the replication fork, in short
stretches of DNA known as Okazaki fragments. This strand is known
as the lagging strand.
 Once replication is completed, the RNA primers are replaced by DNA
nucleotides and the DNA is sealed with DNA ligase, which creates
phosphodiester bonds between the 3'-OH of one end and the 5'
phosphate of the other strand
OPTIONAL ILLUSTRATION
.
Depiction of DNA replication at replication fork. a: template strands, b:
leading strand, c: lagging strand, d: replication fork, e: RNA primer, f:
Okazaki fragment
OPTIONAL
ILLUSTRATION
OPTIONAL
ILLUSTRATION
BASIC SIMILARITY IN MACHINISM
Both are bi-directional processes
DNA polymerases work 5’ to 3’
Leading and lagging strands
Primers are required
Initiator proteins/enzymes bind at
the origin of replication
TERIMA KASIH
(THANKS)
DNA replication in Eukaryotes occurs in three
stages
– Initiation
• Proteins bind to DNA and open up double helix
• Prepare DNA for complementary base pairing
– Elongation
• Proteins connect the correct sequences of
nucleotides into a continuous new strand of DNA
– Termination
• Proteins release the replication complex