REPLICATION
AND
REPAIR
LECTURE OBJECTIVES
At the end of lecture the student should be able to;
• Define replication.
• State the theories of replication
• Deduce the appropriate time for replication in cell cycle.
• List the requirements for replication
• Explain the process of replication
• Justify the need for RNA primer in replication
• Differentiate the process of replication on leading and lagging strand
• Differentiate between DNA polymerase I and III.
• Justify that replication is a flawless process (proofreading activity).
• Explain the processes of repair in DNA replication and relate this with disease processes
like xeroderma pigmentosa.
• Differentiate the replication of DNA in pro and eukaryotes
REPLICATION
Copying of DNA molecule into another DNA molecule
YING AND YANG OF REPLICATION
1.
2.
3.
4.
5.
Semi-conservative
Primer is required
Template is required
Elongation occurs 5’ - 3’
Bidirectional
THEORIES OF REPLICATION
Theory
Conservative
Features
•
•
No change in parent
strand
One new strand is
formed
Semi-conservative
•
•
Two strands are formed
Parent strand
represented in each of
the daughter nuclei
Discontinuous
•
Fragments of new and
old strands are present
in new molecules
CELL CYCLE AND TIME OF REPLICATION
SOME PREREQUISITES
WHY PRIMERS
o
o
o
o
WHY TEMPLATE
Short stretch of
polyribonucleotide (8 – 10
nucleotides)
Formed at DNA
Stabilize first
deoxyribonucleotide
Facilitate DNA polymerase III
Requirements for Replication
 Deoxyribonucleotides
 Adenosine triphophate
 Guanine triphosphate
 Cytidine triphosphate
 Thymidine triphosphate
 ATP
REQUIREMENTS
FOR
REPLICATION
 Enzymes





Primase
Helicase
Polymerase (I,
II, III)
Ligase
Tpoisomerase
SUBSTRATES OF REPLICATION
Proteins
 DnaA
 SSB single
strand binding
proteins
INITIATION






Bidirectional
Away from the origin.
Protein complex Prepriming complex Maintained the strands separated.
Dna A protein  20-50, AT rich, ATP
Single stranded DNA binding proteins polymerase activity, protect from nucleases.
DNA Halicase Two strands unwinds and separate
SITE
SITE
OF
OF
ORIGIN
IN
PROKARYOTES
ORIGIN IN EUKARYOTES
REPLICATION FORK
ELONGATION
LEADING STRAND:
• Direction of helicase 3’ – 5’
• Direction of polymerase 3’ – 5’
• One primer at each point of origin
LAGGING STRAND
•
•
•
•
Direction of helicase 5’ – 3’
Direction of Polymerase 3’ – 5’
More than one primer
DNA synthesized in short stretches Okazaki Fragments
DIFFERENCES
DNA Polymerase I
• Reading 3’ – 5’
•
•
•
•
•
Polymerizing 5’ – 3’
Proof reading 3’ – 5 prime
Incorrect base pair
One at a time
Proofreading 5’ – 3’
• Correct base pair
• Many at a time
DNA Polymerase III
• Reading 3’ – 5’
• Polymerizing 5’ – 3’
• Proof reading 3’ – 5 prime
• Incorrect base pair
• One at a time
REPLICATION IN EUKARYOTES
•
•
•
•
•
Linear DNA
Cell Cycle
DNA polymerases
Telomerase
Reverse transcriptase
•
Inhibition of DNA synthesis by nucleoside analogs
DNA POLYMERASES
Polymerase
Pol 
Function
• Contains Primase
• Initiates DNA
synthesis
Proof Reading

Pol 
Repair
Pol 
Replicates mitochondrial DNA +
Pol 
Elongates leading strands and
okazai fragments
+
Pol 
Repair
+
TELOMERASE

TELOMERASE
•
Extreme 5’ end of lagging strands  Telomers.
•
Non coding sequence of DNA complex with protein.
•
Protect the ends by nucleosidases.
REVERSE TRANSCRIPTASE
 Retroviruses:
 Uses RNA as a template for the synthesis of viral DNA  integrated in host
DNA.
 Reverse transcriptase in human cells
 Telomerase (hTRT)
RNA template complementary to DNA telomer sequence
Reverse transcriptase activity.
INHIBITION OF DNA SYNTHESIS BY NUCLEOSIDE ANALOGS
•
•
•
•
•
DNA chain growth  blocked  Sugar portion of nucleoside.
OH is removed from 3rd C of deoxyribose 2’,3’ dideoxyinosine
Deoxyribose  arabinose
Cytosine arabinoside  anticancer
Adenine arabinoside  anti viral
PALINDROME
•
A palindrome is a word, phrase, or sentence that is spelled identically read either forward
or backward; e.g
– ROTATOR
– NURSES RUN.
•
Regions of DNA with inverted repeats of base sequence having twofold symmetry
over two strands of DNA.
Self-complementary within each strand and therefore have the potential to form hairpin
or cruciform (cross-shaped) structures.
•
PALINDROME
DNA REPAIR
Damage
Time of Cause
Correction
in Cell
Cycle
Enzyme
Involves
Repair
Enzymes
Thymine Dimer
G1
UV rays
Excision
Polymerase
endonuclease Ligase
Cytosine Deamination
G1
SpontaneousChemicals Uracil
GlysylaeAP
endonuclease
ApurinationApyrimidination G1
Spontaneous
Heat
AP
endonuclease
Mismatched Base
Replication Error
Proof
reading
activity
G2
******************************************************************************