DNA replication and repair - University of Leicester · REPLICATION –GENERAL PRINCIPLES START...
Transcript of DNA replication and repair - University of Leicester · REPLICATION –GENERAL PRINCIPLES START...
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BS2009 - GENOMES
DNA replication
and repair
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REPLICATION – GENERAL
PRINCIPLES
START
Must be ready
Must know where to start
FINISH
Must all finish
Must ensure that each piece of DNA is replicated
only once
Therefore must know where to finish
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REPLICATION – GENERAL
PRINCIPLES
ACCURACY/FIDELITY
Proof reading/repair
Must distinguish between original and copy
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S phase
DNA synthesis
G2
Growth phase 2M
Mitosis
G1
Growth phase 1
Go
Quiescent
Main checkpoints
THE EUKARYOTIC CELL CYCLE
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REPLICATION – GENERAL
PRINCIPLES
STARTMust be ready: G1
Must all start at the same time: G1 S
Must know where to start
ORIGIN OF REPLICATION
FINISHMust all finish (complete S)
Must ensure that each piece of DNA is replicated
only once
Therefore must know where to finish (REPLICON)
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REPLICATION – GENERAL
PRINCIPLES
ACCURACY/FIDELITYProof reading/repair (G2)
Must distinguish between original and copy
(EPIGENETICS)
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Summary
DNA Replication
• Occurs during S phase of the cell
cycle
• Semi-conservative (Meselson &
Stahl)
• 5’ → 3’direction
leading strand
lagging strand (discontinuous)
RNA primer
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Summary
• Single origin in bacteria
• Multiple origins in eukaryotes
• Bi-directional
• ARS elements from yeast
cloned origins
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SEMI CONSERVATIVE
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5’3’
5’OH 3’
5’3’
5’
5’3’
5’ OH 3’
OH 3’
DNA polymerase
DNA synthesis always occurs by adding
nucleotides to the 3’-OH of the growing strand.
Synthesis is always in the 5’- 3’ direction
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E. Coli: DnaG
SSB
DnaB
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DnaB
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(SSB)
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Replication Enzymes
• DNA Polymerase - Matches the correct
nucleotides then joins adjacent
nucleotides to each other
• Primase - Provides an RNA primer to
start polymerization
• Ligase - Joins adjacent DNA strands
together (fixes “nicks”)
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• Helicase - Unwinds the DNA and melts it
• Single Strand Binding Proteins - Keep
the DNA single stranded after it has been
melted by helicase
• Gyrase - A topisomerase that Relieves
torsional strain in the DNA molecule
• Telomerase - Finishes off the ends of
DNA strands
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exonuclease
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Eukaryotes vs Prokaryotes• Enzymology, fundamental features,
replication fork geometry, and use of multiprotein machinery conserved
• More protein components in Euk replication machinery
• Replication must proceed through nucleosomes
• Replication fork moves 10X faster in Prok.
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REPLICATION – GENERAL
PRINCIPLES
START
FINISH
ACCURACY/FIDELITY
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Accuracy and Fidelity
Maintenance of DNA
Sequences
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Maintenance of DNA Sequences
DNA Polymerase as Self Correcting Enzyme
• Correct nucleotide has greater affinity for
moving polymerase than incorrect nucleotide
• Exonucleolytic proofreading of DNA
polymerase– DNA molecules w/ mismatched 3’ OH end are not effective
templates; polymerase cannot extend when 3’ OH is not base
paired
– DNA polymerase has separate catalytic site that removes
unpaired residues at terminus
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Maintenance of DNA SequencesDNA Polymerase as Self Correcting Enzyme
Two catalytic sites
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Strand Directed Mismatch Repair System
• Removes replication errors not recognized by replication machine
• Detects distortion in DNA helix
• Distinguishes newly replicated strand from parental strand by methylation of A residues in GATC in bacteria
• Methylation occurs shortly after replication occurs
• Reduces error rate 100X
• 3 Step Process
recognition of mismatch
excision of segment of DNA containing mismatch
resynthesis of excised fragment
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Strand Directed Mismatch
Repair in Mammals
• Newly synthesized strand is
preferentially nicked and can be
distinguish in this manner from
parental strand
• Defective copy of mismatch repair
gene predisposed to cancer
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Strand Directed Mismatch Repair System
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Causes of DNA Damage
•Chemical mutagens
•Radiation
•Free radicals
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RADIATION = ENERGY
ENERGY DEPOSITION IN DNA
DNA DAMAGE
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S-A…….T-S
P P
S-A…….T-S
P P
S-T…… A-S
P P
S-G……C-S
P P
S-C….....G-S
P P
S-T……..A-S
P P
S-A……..T-S
P P
P
S
P
S-G
P
S-C
P
S-T
P
S-A……..T-S
P P
S-A…….T-S
P P
S-A…….T-S
P
A-S
P
C-S
P
G-S
P
S
P
RADIOLYSIS OF WATER
H2O OH + H + e-. .
OH
OH
.
.
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Carbohydrate + O2
Energy + CO2 + H2O
RESPIRATION AND AGEING
02 H2O2 OH. .
DNA damage
~10000 lesions/cell/day
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DNA RepairTypes of DNA Damage: Base Loss and Base Modification
DepurinationChemical Modification Photodamage thymine dimer
Chemical Modification by O2 free radicalsDeamination
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DNA Repair
►Despite 1000’s of alterations that occur in
DNA each day, few are retained as mutations
►Efficient repair mechanisms
►Importance of DNA repair highlighted by:
Number of genes devoted to DNA repair
mutation rates with inactivation or loss of DNA
repair gene
►Defects in DNA repair associated with
several disease states
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DNA replication and repair
disordersDisorder Frequency Defect
Fanconi’s anaemia 1/22,000 in some
popns.
Deficient excision
repair
Hereditary nonpolyposis
colon cance
1/200 Deficient mismatch
repair
Werner’s syndrome 3/1,000,000 Deficient helicase
Xeroderma pigmentosum 1/250,000 Deficient excision
repair
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DNA Repair
DNA Damage Can Activate Expression of
Whole Sets of Genes
• Heat Shock Response
• SOS Response
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DNA Repair
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DNA Repair
Base Excision Repair
a. DNA glycosylase recognizes damaged base
b. Removes base leaving deoxyribose sugar
c. AP endonuclease cuts phosphodiester backbone
d. DNA polymerase replaces missing nucleotide
e. DNA ligase seals nick
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Failure of DNA repair
• When DNA repair fails, fewer mutations corrected increase in
number of mutations in the genome.
• The protein p53 monitors repair of damaged DNA.
• If damage too severe, p53 protein promotes programmed cell
death (apoptosis)
• Mutations in genes encoding DNA repair proteins can be inherited
overall increase in mutations as errors or damage to DNA no
longer repaired efficiently.
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DNA DAMAGE
SURVEILLANCE
RECOGNITION
SIGNALLING
REPAIR