bs2009 - genomes
DESCRIPTION
BS2009 - GENOMES. DNA replication and repair. 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. REPLICATION – GENERAL PRINCIPLES - PowerPoint PPT PresentationTRANSCRIPT
![Page 1: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/1.jpg)
BS2009 - GENOMES
DNA replication and repair
![Page 2: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/2.jpg)
REPLICATION – GENERAL PRINCIPLES
STARTMust be ready
Must know where to start
FINISHMust all finish
Must ensure that each piece of DNA is replicated only once
Therefore must know where to finish
![Page 3: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/3.jpg)
REPLICATION – GENERAL PRINCIPLES
ACCURACY/FIDELITYProof reading/repair
Must distinguish between original and copy
![Page 4: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/4.jpg)
S phaseDNA synthesis
G2Growth phase 2M
Mitosis
G1Growth phase 1
GoQuiescent
Main checkpoints
THE EUKARYOTIC CELL CYCLE
![Page 5: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/5.jpg)
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)
![Page 6: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/6.jpg)
REPLICATION – GENERAL PRINCIPLES
ACCURACY/FIDELITYProof reading/repair (G2)
Must distinguish between original and copy (EPIGENETICS)
![Page 7: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/7.jpg)
SummaryDNA Replication• Occurs during S phase of the cell
cycle• Semi-conservative (Meselson &
Stahl)• 5’ → 3’direction
leading strandlagging strand (discontinuous)RNA primer
![Page 8: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/8.jpg)
Summary
• Single origin in bacteria• Multiple origins in eukaryotes• Bi-directional• ARS elements from yeast
cloned origins
![Page 9: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/9.jpg)
SEMI CONSERVATIVE
![Page 10: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/10.jpg)
5’3’
5’ OH 3’
5’3’
5’
5’3’
5’ OH 3’
OH 3’
DNA polymerase
DNA synthesis always occurs by addingnucleotides to the 3’-OH of the growing strand.
Synthesis is always in the 5’- 3’ direction
![Page 11: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/11.jpg)
![Page 12: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/12.jpg)
E. Coli: DnaG
SSB
DnaB
![Page 13: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/13.jpg)
DnaB
![Page 14: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/14.jpg)
![Page 15: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/15.jpg)
(SSB)
![Page 16: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/16.jpg)
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”)
![Page 17: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/17.jpg)
• 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
![Page 18: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/18.jpg)
![Page 19: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/19.jpg)
![Page 20: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/20.jpg)
exonuclease
![Page 21: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/21.jpg)
![Page 22: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/22.jpg)
![Page 23: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/23.jpg)
![Page 24: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/24.jpg)
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.
![Page 25: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/25.jpg)
REPLICATION – GENERAL PRINCIPLES
START
FINISH
ACCURACY/FIDELITY
![Page 26: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/26.jpg)
Accuracy and Fidelity
Maintenance of DNA Sequences
![Page 27: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/27.jpg)
Maintenance of DNA SequencesDNA 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
![Page 28: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/28.jpg)
Maintenance of DNA SequencesDNA Polymerase as Self Correcting EnzymeTwo catalytic sites
![Page 29: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/29.jpg)
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 mismatchexcision of segment of DNA containing mismatchresynthesis of excised fragment
![Page 30: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/30.jpg)
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
![Page 31: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/31.jpg)
Strand Directed Mismatch Repair System
![Page 32: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/32.jpg)
Causes of DNA Damage
• Chemical mutagens• Radiation• Free radicals
![Page 33: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/33.jpg)
RADIATION = ENERGY
ENERGY DEPOSITION IN DNA
DNA DAMAGE
![Page 34: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/34.jpg)
![Page 35: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/35.jpg)
S-A…….T-SP P S-A…….T-SP P S-T…… A-SP P S-G……C-SP P S-C….....G-SP P S-T……..A-SP P S-A……..T-SP P
P S P S-GP S-CP S-TP S-A……..T-SP P
S-A…….T-SP 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
.
.
![Page 36: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/36.jpg)
Carbohydrate + O2
Energy + CO2 + H2O
RESPIRATION AND AGEING
02 H2O2 OH. .DNA damage
~10000 lesions/cell/day
![Page 37: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/37.jpg)
DNA RepairTypes of DNA Damage: Base Loss and Base Modification
DepurinationChemical Modification Photodamage thymine dimer
Chemical Modification by O2 free radicalsDeamination
![Page 38: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/38.jpg)
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
![Page 39: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/39.jpg)
DNA replication and repair disorders
Disorder Frequency DefectFanconi’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
![Page 40: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/40.jpg)
DNA RepairDNA Damage Can Activate Expression of
Whole Sets of Genes• Heat Shock Response• SOS Response
![Page 41: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/41.jpg)
DNA Repair
![Page 42: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/42.jpg)
DNA RepairBase Excision Repaira. DNA glycosylase recognizes
damaged baseb. Removes base leaving
deoxyribose sugarc. AP endonuclease cuts
phosphodiester backboned. DNA polymerase replaces
missing nucleotidee. DNA ligase seals nick
![Page 43: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/43.jpg)
![Page 44: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/44.jpg)
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.
![Page 45: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/45.jpg)
![Page 46: BS2009 - GENOMES](https://reader035.vdocuments.site/reader035/viewer/2022062222/56815c46550346895dca4546/html5/thumbnails/46.jpg)
DNA DAMAGE
SURVEILLANCE
RECOGNITION
SIGNALLING
REPAIR