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INTRODUCTION
Nuclease
hydrolyse the phosphodiester bonds
exonuclease
endonuclease
Polymerase or replicase
catalyse the formation of polymers
Prokaryotic DNA replication
RAW MATERIALS
ENZYMES
TEMPLATES
RNA PRIMERS
REPLICONS
REPLISOMES
PRIMOSOMES
oENZYMES
DNA polymerase (I, II, III)
Topoisomerase/ gyrase
SSBP
DNA polymerase I
Functions:
1. Template site for binding template DNA
2. Primer site to bind primer RNA
3. Used to fill gap between Okazaki fragments that are formed during lagging
strand synthesis.
4. Catalyse DNA repair and discontinuous DNA synthesis.
5. Dna Repair.
DNA polymerase II
90KD Polypeptide
Coded by PolB gene
Functions:
1. Assist in polymerisation
2. Mainly involved in DNA repair
DNA polymerase III
. synthesizes base pairs at a rate of around 1000 nucleotides per
second
. Complex molecule
. Act as 5 3 and 3 5 exonuclease
. Formed of 10 subunits
Functions:
1. Chain elongation in leading strand
2. Essential for in vivo DNA replication
3. Help in repair
DNA helicase/ Dna B protein
Involved in strand separation
ATP dependent enzyme
2 types
> Pri A protein
> Rep protein
Pri A protein
or helicase II and III
Moves on 5 3
Attaches to the template for the lagging strand
Rep protein
Direct leading strand synthesis
Moves on 3 5
In rolling circle replication
Topoisomerase or gyrase
Causes topological changes in DNA
Based on whether they cause single strand or double strand
break it is of 2 types
RNA PRIMER
Short oligonucleotide to start replication
Produced with the help of DNA primase/RNA polymerase
Hydrogen bonded to DNA
o REPLICONS
Requires
DnaB - Unwinding
primase - primer addition
DNA pol III - elongation
SSBP - prevent rewinding
RNAse H - removes RNA primer
DNA pol I - fill the gap
DNA ligase - join the okazaki fragments
Discontinous synthesis of lagging Continuous synthesis of leading
strand strand
Multiple primer needed Need only one primer
Continues bi-directionally
until they meet
Rolling Circle Replication
begins with a nick (single-stranded break) at the origin
The 5 end is displaced from the strand
3 end acts as a primer for DNA polymerase III, which
synthesizes a continuous strand
The 5 end continues to be displaced as the circle rolls, and is
protected by SSBs until discontinuous DNA synthesis makes it a
dsDNA again
During viral assembly it is cut into individual viral chromosomes
and packaged into phage head.
Eukaryotic DNA replication
not as well understood as bacterial replication
more complex
Large linear chromosomes
Topoisomerase
2 types
Class I ( Topo I & Topo III)
SSBP
Human SSBP or RP-A
Tetramer
Origins of Replication
origins of replication found in eukaryotes have some similarities
to those of bacteria
They are 100-150 bp in length
They have a high percentage of A and T
They have three or four copies of a specific sequence
Similar to the bacterial DnaA boxes
Origin recognition complex (ORC)
A six-subunit complex that acts as the initiator of eukaryotic
DNA replication
It appears to be found in all eukaryotes
Requires ATP to bind ARS elements
Single-stranded DNA stimulates ORC to hydrolyze ATP
Initiation
Multiple origin
Histones associated with DNA should be removed
Rate 105 bp/min
Takes 1000 times more replication time than that of prokaryotic
replication
Large amount of DNA in chromosome at multiple replisomes
Initiative protein selects the origin and activates it with the help of
other proteins
Initiatve protein is ORC
Y shaped intermediate will form
Elongation
Primer excised by endonuclease or RNaseH
Nucleosome assembly
Termination
Occurs by telomere replication
Telomeric sequences consist of
Moderately repetitive tandem arrays
3 overhang that is 12-16 nucleotides long
Telomeric sequences typically consist of
Several guanine nucleotides
Often many thymine nucleotides
DNA polymerases possess two unusual features
1. They synthesize DNA only in the 5 to 3 direction
2. They cannot initiate DNA synthesis
These two features pose a problem at the 3 end of linear chromosomes
Therefore if this problem is not solved
The linear chromosome becomes progressively shorter with
each round of DNA replication
Indeed, the cell solves this problem by adding DNA sequences to
the ends of telomeres
This requires a specialized mechanism catalyzed by the enzyme
telomerase
Telomerase contains protein and RNA
The RNA is complementary to the DNA sequence found in
the telomeric repeat
This allows the telomerase to bind to the 3 overhang
Step 1 = Binding
The binding-
polymerization- Step 2 = Polymerization
translocation cycle can
occurs many times
The complementary
strand is made by primase,
DNA polymerase and ligase
RNA primer
http://www.bioteach.ubc.ca/TeachingResources/MolecularBiology/DNAReplicat
ion.swf