DNA REPLICATION I - MOLEKÜLCEgenetic continuity between parental and progeny cells is maintained by semiconservative replication of dna, as predicted by the watson–crick model dna

DNA REPLICATION I

molekulce.com/Tuba ERTÜRK

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




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


3 POSSIBLE MODELS

OF DNA REPLICATION

ARE POSSIBLE:

– CONSERVATIVE

– SEMICONSERVATIVE

– DISPERSIVE


The Meselson-Stahl experiment

demonstrated that:

DNA replication is semiconservative

each new DNA molecule consists of

one old strand and one newly

synthesized strand



1 The Taylor-Woods

Hughes experiment

demonstrated that

DNA

replication is

semiconservative in

eukaryotes


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


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


Unwinding DNA Helix

DnaA binds to the

origin of replication

(oriC) and is

responsible for the

initial steps in

unwinding the helix


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


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


Both DNA strands are synthesized concurrently

by looping the lagging strand to invert the

physical but not biological direction of synthesis


DNA Replication is Uncommonly Accurate


Proofreading and

error correction are

integral parts of DNA

replication

All of the DNA

polymerases have 3'

to 5’ exonuclease

activity that allows

proofreading


Circular DNA Replication


DNA Replication in bacteria


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


E. coli OriC


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


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


Dna A proteins bind to DnaA sequences

DnaA proteins unfold the

AT-rich regions


BIDIRECTIONAL REPLICATION


Initiator Proteins at ORI


Initiation in E. coli


ALL THE MOLECULES AND PROTEINS

ATTACHED AND TAKE PART IN THE

REPLICATION PROCESS FORM A COMPLEX

CALLED AS REPLISOME


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


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


DNA Helicase Separates Strands


Helicase Binds to DNA Polymerase III


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


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)


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


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


Helicase – Primase (Primosome)


DNA POLYMERASE

DNA POLYMERASE RESEMBLES A CLOSED RIGHT

HAND FIGURE

IT HAS 3 DOMAINS;

1- Thumb

2- Palm

3- Fingers


Various DNA Polymerases


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


CHAIN ELONGATION OCCURS IN THE 5' TO 3’

DIRECTION BY ADDITION OF ONE NUCLEOTIDE AT

A TIME TO THE 3' END


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


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


DNA polymerase III has 10 subunits


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


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


Sliding DNA Clamp


Sliding DNA Clamp Increases Processivity


E. coli dimer sliding clamp Human trimer sliding clamp

Subunit PCNA


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




DNA synthesis at a single replication fork


How DNA polymerases are kept attached to

replication fork during the synthesis of leading and

lagging DNA strands?

Trombone Model


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


Periodically, DNA primase

interacts with DNA helicase

RNA primer is synthesized on

the lagging strand



When an Okazaki fragment

is completed on the lagging

strand DNA polymerase is

released from the sliding

clamp



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


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

Documents

DNA REPLICATION I - MOLEKÜLCEgenetic continuity between parental and progeny cells is maintained by semiconservative replication of dna, as predicted by the watson–crick model dna