DNA replication: The process of duplication of the entire genome prior to cell division.
Occurs only once in a cell cycle - at S phase
Salient features of DNA replication
DNA replication is a
Semiconservativeprocess–Each copy of DNA has one parental strand and one newly synthesized strand.
Template directed process - Template strand is required – newly synthesized strand is complementary to the template
strand.
The template strand is read from 3’-5’ direction and the new strand is synthesized from 5’-3’direction
Primerdependentprocess:-RNAprimeris required to start DNA synthesis
Semi discontinuous process:- One strand is synthesized continuously–[Link] short
fragments (discontinuously) – lagging strand
Bidirectional process
Accurate process:- High rate of accuracy is due to fidelity of base selectionand proofreadingactivity of DNA polymerase as
well as mismatch repairmechanism.
The processis catalyzedbya magnesiumdependentenzyme DNA polymerase. Nascent strand is synthesized in 5’-3’ direction.
Replicationtakesplacein3phases–Initiation,elongationand termination
Initiation:-
Replication begins at a specific site called origin of replication (ori) in prokaryotes. In [Link] it is called oriC . In Bacteriophage λ it is referred
to as oriλ. Ori C is made of 2 regions – a) Multiple short repeated sequences to bind specific initiator protein and b) AT rich region where initial
unwindingoccurs.
DnaA recognizes the multiple short repeat sequence in oriC and binds to the DNA. This causes a conformational chane in the DNA and the
DNA unwinds in the AT rich region. Two Dna B proteins (helicase) loads onto the single stranded DNA with the help of the helicase loader
protein Dna C. The ssDNA are bound by Single strandbindingproteinswhichpreventsthereannealingoftheDNA. Thus replication bubble
isformed. Ineukaryotes there are multiple sites for origin of replication. In yeast these are called as ARS (Autonomously Replicating
Sequences).
The proteins that take part in initiation in eukaryotes are :-
Bacteria Yeast
Origin site OriC ARS
Initiator protein Dna A ORC (Origin recognition complex)
Helicase Dna B MCM (Minichromosome maintenance rotein)
Helicase loader Dna C Cdc6p (celldivisioncycle 6 protein)
Elongation:-
• Each replication bubble has two replication forksand synthesis of DNA proceedsbidirectionally.
• Elongation phase involves twoprocesses:
Leading strand synthesis
Lagging strand synthesis
Replication fork:-
In each replication fork the following are seen
Helicase:
• BindstosinglestrandedDNAinreplicationbubbleandcauses progressive unwinding of DNA
• ATPdependentprocess Topoisomerase
• Moves ahead of the replication fork and removes the supercoiling produced duringunwinding
Single-strand DNA-binding protein (SSB)
• These bind to unwound DNA produced as a result of helicase activity
• Their function isto stabilize the ssDNA and prevent their reannealing
• TheeukaryoticSSBiscalledReplicationproteinA. Primase
• Primase synthesizes primer
• DNA polymerase can add dNTP only to the 3’end of existing [Link] first
nucleotide, hence a primer is required.
• Primase is anRNA polymerase which catalyses the synthesis of RNA primer complementary to the template strand
Primer
• Primer is 10-100 nucleotides longRNA
• Primer is synthesized in 5’-3’direction
• Laterthe primerisremoved and replaced by DNA bygapfilling DNA polymerase
• IneukaryotesDNApolymeraseαitself hasprimaseactivity Leading strand
• DNApolymerase:2DNApolymerasesbindtothereplication fork and start synthesis of the nascent strand.
• Both the strands are replicatedsimultaneously
• Inreplicationfork,directionof unwindingisfavourableforthe synthesis of one strand, which is synthesized continuously. This is
called the leadingstrand
Lagging strand
• The othertemplate strand is copied in shortfragments – okazaki fragments.
• 100-250 nucleotides long each with RNA primer of about 10 nucleotides in length
• RnaseHremovestheprimer
• DNApolymerasefillsthegap
• DNA ligase seals the gap
DNA polymerase
• Multi subunit, magnesium dependentprotein.
It has 3 importantproperties
Polymerase activity
Proof reading activity
Processivity Polymerase activity
• Catalysesthe polymerisationofdNTP to 3’-end ofa nascent strand.
• Nascent strand is elongated from 5’-3’ end
• Itrequiresa Primer:DNApolymerase canadddNTP only tothe 3’end of existing nascent strand.
• It requires a template
• Base selection – The appropriate base is selected by obeying Watson- Crick base pairrule.
• Proof reading activity: 3’-5’ exonuclease
• DNA polymerase has 3’ exonuclease activity. If a wrong base is added- the enzyme does not move forward – it removes the wrong
base by exonuclease activity, adds the right base and then proceeds down.
Processivity
• DNA polymerase adds many dNTPs to the nascent strand before dissociating from it. This increases the speed of
replication process. It is due to the presence of Beta sliding clamp.
Types of DNA polymerase in prokaryotes
Types Functions
DNA polymerase I Gap filling in replication, repair and
DNA polymerase II recombination
DNA repair
DNA polymerase III Replication
Types of DNA polymerase ineukaryotes
Types Functions
DNA polymerase α Primase activity
DNA polymerase β DNA repair
DNA polymerase γ MitochondrialDNAsynthesis
DNA polymerase δ Laggingandleading strand
DNA polymerase ε synthesis
Repair
The proteins that take part in elongation in eukaryotes are:-
Prokaryotes Eukaryotes
Beta clamp PCNA (Proliferating cell nuclear antigen)
Clamp loader Replication factor C
Single strand binding proteins Replication protein A
Primase DNA Polymerase alpha
Termination Prokaryotes
Eventually,the two replication forksof the circular [Link] chromosome meet at a terminus region called
Ter. The Ter sequences function as binding sites for the protein Tus (terminusutilizationsubstance).The Tus-Ter complexcanarresta
[Link],wheneitherreplicationfork encounters a functional Tus-Ter complex, it halts. Formimg two
topologically interlinked (catenated) circular chromosomes – catenanes. These are separated by catenase (DNA topoisomerase IV).
In Eukaryotes:-
• EndsofDNA doesnot have genes– made of repetitive sequences called telomeres
• Telomeres consist of short TGrepeats.
• Human telomeres have variable number of 5’-TTAGGG-3’ repeats repeated several thousand times
• With each cell division, the telomere shortens.
• Length of telomere – indicator of the age of the cell
• Senescent cell telomeres reach a critical length beyond this no cell division occurs
Telomerase:-
Length of telomere is maintained by telomerase
Telomeraseisaribonucleoproteincomplex–RNAandprotein.
RNAactsastemplateand proteinsynthesizesthe telomere– This enzyme is an RNA dependent DNA polymerase.
Telomerase is active in dividingcells
Differentiated cells do not expresstelomerase
In malignant cells telomerase isactivated
In eukaryotes sfter replication, the two copies of genome are organised into nucleosomes. Histones are synthesized in G1 phase. The
histones of the parent DNA is equally shared [Link] into nucleosomes using 50%
of parental histones and 50% of newly synthesized histones