DNA REPLICATION I
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GENETIC CONTINUITY
BETWEEN PARENTAL AND
PROGENY CELLS IS
MAINTAINED BY
SEMICONSERVATIVE
REPLICATION OF DNA, AS
PREDICTED BY THE
WATSON–CRICK MODEL
DNA SYNTHESIS IS SIMILAR
IN PROKARYOTES AND
EUKARYOTES, BUT MORE
COMPLEX IN EUKARYOTES molekulce.com/Tuba ERTÜRK
DNA SYNTHESIS IS A COMPLEX BUT ORDERLY
PROCESS,OCCURRING UNDER THE DIRECTION OF
A MYRIAD OF ENZYMES AND OTHER PROTEINS
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DNA IS REPRODUCED BY
SEMI-CONSERVATIVE
REPLICATION
THE COMPLEMENTARITY
OF DNA STRANDS
ALLOWS EACH STRAND
TO SERVE AS A TEMPLATE
FOR SYNTHESIS OF THE
OTHER
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3 POSSIBLE MODELS
OF DNA REPLICATION
ARE POSSIBLE:
– CONSERVATIVE
– SEMICONSERVATIVE
– DISPERSIVE
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The Meselson-Stahl experiment
demonstrated that:
DNA replication is semiconservative
each new DNA molecule consists of
one old strand and one newly
synthesized strand
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1 The Taylor-Woods
Hughes experiment
demonstrated that
DNA
replication is
semiconservative in
eukaryotes
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DNA REPLICATION BEGINS
AT THE ORIGIN OF
REPLICATION AND IS
BIDIRECTIONAL RATHER
THAN UNIDIRECTIONAL
A REPLICON IS THE LENGTH
OF DNA THAT IS
REPLICATED FOLLOWING
ONE INITIATION EVENT AT A
SINGLE ORIGIN
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7 KEY ISSUES THAT MUST BE RESOLVED
DURING DNA REPLICATION
– unwinding of the helix
– reducing increased coiling generated during
unwinding
– synthesis of a primer for initiation
– discontinuous synthesis of the second strand
– removal of the RNA primers
– joining of the gap-filling DNA to the adjacent
strand
– proofreading
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Unwinding DNA Helix
DnaA binds to the
origin of replication
(oriC) and is
responsible for the
initial steps in
unwinding the helix
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To elongate a polynucleotide chain, DNA
polymerase III requires a primer with a free 3’OH
group
Enzyme primase synthesizes an RNA primer
that provides the free 3'-OH required by DNA
polymerase III
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As replication fork moves,
only 1 strand can serve as
template for continuous
DNA synthesis—the
leading strand
Opposite lagging strand
undergoes
discontinuous DNA
synthesis
At the end of replication
Okazaki fragments are
joined by the activity DNA
ligase enzyme
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Both DNA strands are synthesized concurrently
by looping the lagging strand to invert the
physical but not biological direction of synthesis
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DNA Replication is Uncommonly Accurate
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Proofreading and
error correction are
integral parts of DNA
replication
All of the DNA
polymerases have 3'
to 5’ exonuclease
activity that allows
proofreading
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Circular DNA Replication
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DNA Replication in bacteria
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Origin of replication in E. coli is named as oriC
– Origin Chromosomal replication
THERE ARE THREE IMPORTANT DNA
SEQUENCES WERE DEFINED IN oriC REGION;
– AT- rich region
– DnaA boxes
– GATC methylation sites
ORIGIN OF REPLICATION IN E. coli
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E. coli OriC
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REPLICATOR
SPECIFIC SEQUENCES RESPONSIBLE FOR
THE INITIATION OF THE REPLICATION
PROCESS- REPLICATION ORIGIN SITES
THEY HAVE SPECIFIC BINDING REGIONS FOR
THE INITIATOR PROTEIN
THEY HAVE LOOSE A-T RICH REGIONS
THESE SPECIFIC SEQUENCES ARE SIMILAR
IN MOST OF THE ORGANISMS RESEMBLING A
TYPICAL REPLICATOR MOTIF
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INITIATOR (DNA-A PROTEIN)
RECOGNIZES THE SPECIFIC SEQUENCES IN
REPLICATOR SITES ON DNA AND INITIATES
THE REPLICATION
THERE ARE 3 ROLES OF INITIATOR PROTEINS:
1. BINDING TO THE REPLICATOR
2. UNFOLDING OF THE DNA AT THE BINDING
SITE
3. GATHERING OF THE ASSOCIATED
MOLECULES AND PROTEINS TO THE
REPLICATION SITE
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Dna A proteins bind to DnaA sequences
DnaA proteins unfold the
AT-rich regions
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BIDIRECTIONAL REPLICATION
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Initiator Proteins at ORI
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Initiation in E. coli
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ALL THE MOLECULES AND PROTEINS
ATTACHED AND TAKE PART IN THE
REPLICATION PROCESS FORM A COMPLEX
CALLED AS REPLISOME
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Replisome complex
5 3 5
3
3 5
5
Helicase
Topoisomerase
Single strand DNA-binding
Proteins (SSBPs)
DNA
polimerase RNA
primer
Leading strand
Lagging strand RNA
primer
Primase
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DNA HELICASE
DNA HELICASE CATALYZES THE UNWINDING OF
THE DNA STRANDS BY USING ATP ENERGY
IT ATTACHES TO ONE OF THE SINGLE STRANDS
HAS AN HEXAMERIC STRUCTURE
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DNA Helicase Separates Strands
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Helicase Binds to DNA Polymerase III
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SSBP: SINGLE STRANDED BINDING PROTEINS
SSBPs ATTACH TO THE NEWLY UNWINDED DNA
STRANDS IN ORDER TO INHIBIT THEIR
REASSOCIATION
SSBPs BINDING IS NOT ASSOCIATED WITH ANY
SEQUENCE RECOGNITION
SSBPs BIND TO ssDNA BY THE ELECTROSTATIC
INTERACTIONS WITH THE PHOSPHATE GROUPS
ON DNA,WHICH IS ALSO A KIND OF COOPERATIVE
BINDING
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DNA TOPOISOMERASES
AS THE dsDNA UNWINDS BY THE ACTIVITY OF
HELICASE THE REMAINING dsDNA IN FRONT
OF THE REPLICATION FORK FORMS A
POSITIVE SUPER-COILING
THE TENSION ON THE SUPERCOILING SITE IS
RELEASED BY THE ACTIVITY OF A
TOPOISOMERASE ENZYME CALLED DNA
GYRASE ENZYME (ATP ENERGY)
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RNA PRIMER
WHEN THE DNA HELIX IS UNWINDED, A SMALL RNA
SEQUENCE (5-10 NUCLEOTIDS) -THE PRIMER-
IS SYNTHESIZED BY THE ACTIVITY OF RNA
PRIMASE ENZYME IN ORDER TO PROVIDE THE FREE
3’-OH GROUP FOR DNA-POLYMERASE FOR
POLYMERIZATION
BECAUSE PRIMASE IS AN RNA-POLYMERASE,
IT DOESN’T NEED A FREE 3’-OH GROUP FOR
POLYMERIZATION
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RNA PRIMER
JUST LIKE HELICASE, PRIMASE IS ACTIVATED BY
THE OTHER PROTEINS OF THE REPLICATION
COMPLEX AND STARTS TO SYNTHESIZE PRIMER
AS THE REPLICATION INITIATES
WHEN THE REPLICATION COMPLETES, PRIMER IS
EXCISED BY DNA POLYMERASE I AND THE GAP IS
FILLED
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Helicase – Primase (Primosome)
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DNA POLYMERASE
DNA POLYMERASE RESEMBLES A CLOSED RIGHT
HAND FIGURE
IT HAS 3 DOMAINS;
1- Thumb
2- Palm
3- Fingers
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Various DNA Polymerases
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DNA Synthesis in Bacteria Involves Five
Polymerases, as well as Other Enzymes
DNA POLYMERASE I CATALYZES DNA SYNTHESIS
AND REQUIRES A DNA TEMPLATE AND ALL FOUR
dNTPS
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CHAIN ELONGATION OCCURS IN THE 5' TO 3’
DIRECTION BY ADDITION OF ONE NUCLEOTIDE AT
A TIME TO THE 3' END
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DNA polymerases I, II, and III CAN
ELONGATE AN EXISTING DNA STRAND
(CALLED A PRIMER) BUT CANNOT
INITIATE DNA SYNTHESIS
ALL THREE POSSESS 3' TO 5‘
EXONUCLEASE ACTIVITY BUT, ONLY
DNA POLYMERASE I DEMONSTRATES 5‘
TO 3' EXONUCLEASE ACTIVITY
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DNA polymerase III,
IS THE ENZYME RESPONSIBLE FOR THE 5' TO 3’
POLYMERIZATION ESSENTIAL in vivo
ITS 3' TO 5' EXONUCLEASE ACTIVITY ALLOWS
PROOFREADING
DNA Polymerase I IS BELIEVED TO BE
RESPONSIBLE FOR:
– REMOVING THE PRIMER
– FILLS GAPS PRODUCED DURING
REPLICATION
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DNA polymerase III has 10 subunits
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holoenzyme
Pol III* molekulce.com/Tuba ERTÜRK
Holoenzyme: (active form) a dimer which consists of 10
different polipeptide chains
Core enzyme: the biggest sub-unit; α,ε, θ units are
attached, catalyzes the polymerization of the
polynucleotide chain and proof-reading
γ (gamma) complex: has 5 sub-units, loading of the
enzyme on to DNA and has clamp loader activity
β (beta) sub-unit: facilates the attachment of the core
enzyme to the DNA strand and forms the sliding clamp
structure
(pi) sub-unit: facilates the attachment of the 2 core
polymerases on the replication fork
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SLIDING CLAMPS
DNA POLYMERASE III CAN SYNTHESIZE 20-100
BASE PAIRS WITHOUT LEAVING THE DNA
TEMPLATE STRAND
SLIDING CLAMPS ARE THE PROTEINS WHICH
ATTACHED TO DNA POLYMERASE ON THE
REPLICATION FORK IN ORDER TO INCREASE THE
PROCESSIVITY OF DNA POLYMERASE
THEY HAVE DIFFERENT SUB-UNITS
AND AN OPEN CENTER TO HOLD DNA
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Sliding DNA Clamp
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Sliding DNA Clamp Increases Processivity
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E. coli dimer sliding clamp Human trimer sliding clamp
Subunit PCNA
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SLIDING CLAMP LOADERS
SLIDING CLAMPS ARE CLOSED CIRCLES AND AN
ADDITIONAL PROCESS IS REQUIRED IN ORDER TO
FACILATE THEIR ATTACHMENT TO DNA
THIS IS DONE BY THE ACTIVITY OF SLIDING
CLAMP LOADERS BY USING ATP ENERGY
AT THE END OF THE REPLICATION SAME
MECHANISM IS USED TO RELEASE OF THE
SLIDING CLAMP FROM THE DNA
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DNA synthesis at a single replication fork
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How DNA polymerases are kept attached to
replication fork during the synthesis of leading and
lagging DNA strands?
Trombone Model
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Trombone Model of DNA Replication in E. coli
Holoenzyme interacts
with helicase with its
-sub unit and attaches to
both of the polymerases
at the same time
One polymerase
replicates the leading
strand and the other
replicates the lagging
strand
SSBPs attach to the
ssDNA molekulce.com/Tuba ERTÜRK
Trombone Model of DNA Replication in E. coli
Periodically, DNA primase
interacts with DNA helicase
RNA primer is synthesized on
the lagging strand
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Trombone Model of DNA Replication in E. coli
When an Okazaki fragment
is completed on the lagging
strand DNA polymerase is
released from the sliding
clamp
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Trombone Model of DNA Replication in E. coli
The new primer attached
lagging strand is targeted
by the sliding clamp loader
and primer-DNA complex
is attached to the sliding
clamp molekulce.com/Tuba ERTÜRK
Trombone Model of DNA Replication in E. coli
Primer-DNA complex is
attached to the sliding clamp
and polymerase on the lagging
strand
Synthesis of the new Okazaki
fragment starts molekulce.com/Tuba ERTÜRK
Termination of replication of the circular chromosome
GAPS ON DNA ARE FILLED WITH THE
ACTIVITIES OF THE DNA POLYMERASE AND
DNA LIGASE ENZYMES molekulce.com/Tuba ERTÜRK