BS2009 - GENOMES DNA replication and repair REPLICATION

BS2009 - GENOMES DNA replication and repair REPLICATION

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 ACCURACY/FIDELITY Proof reading/repair Must distinguish between original and copy THE EUKARYOTIC CELL CYCLE G1 Growth phase 1

Go Quiescent S phase DNA synthesis Main checkpoints M

Mitosis G2 Growth phase 2 REPLICATION GENERAL PRINCIPLES START Must be ready: G1

Must all start at the same time: G1 Must know where to start ORIGIN OF REPLICATION FINISH Must all finish (complete S) Must ensure that each piece of DNA is replicated only once

Therefore must know where to finish (REPLICON) S REPLICATION GENERAL PRINCIPLES ACCURACY/FIDELITY Proof reading/repair (G2)

Must distinguish between original and copy (EPIGENETICS) Summary DNA Replication Occurs during S phase of the cell cycle Semi-conservative (Meselson & Stahl)

5 3direction leading strand lagging strand (discontinuous) RNA primer Summary Single origin in bacteria Multiple origins in eukaryotes Bi-directional

ARS elements from yeast cloned origins SEMI CONSERVATIVE DNA polymerase 5 OH 3

5 3 5 OH 3 5

3 5 3 OH 3 5

DNA synthesis always occurs by adding nucleotides to the 3-OH of the growing strand. Synthesis is always in the 5- 3 direction SSB DnaB E. Coli: DnaG

DnaB (SSB) 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) 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 exonuclease 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. REPLICATION GENERAL PRINCIPLES START FINISH ACCURACY/FIDELITY Accuracy and Fidelity

Maintenance of DNA Sequences 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 Maintenance of DNA Sequences DNA Polymerase as Self Correcting Enzyme Two catalytic sites

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 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 Strand Directed Mismatch Repair System Causes of DNA

Damage Chemical mutagens Radiation Free radicals RADIATION = ENERGY ENERGY DEPOSITION IN DNA DNA DAMAGE

RADIOLYSIS OF WATER . . OH + H + e- H2O

. OH S-A.T-S P S-A.T-S S-A.T-S

P P P S-A.T-S P

P P S-T A-S P P S-GC-S

P P S-C.....G-S P P S-T..A-S P

P S-A..T-S P P . OH

S P S-G P S-C P A-S P

C-S P G-S P S P P S-T

P S-A..T-S P P RESPIRATION AND AGEING Carbohydrate + O2

Energy + CO2 + H2O . 02 H2O2

. OH DNA damage ~10000 lesions/cell/day DNA Repair Types of DNA Damage: Base Loss and Base Modification

Chemical Modification Deamination Depurination Photodamage thymine dimer

Chemical Modification by O2 free radicals DNA Repair Despite 1000s 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 DNA replication and repair

disorders Disorder Fanconis anaemia Hereditary nonpolyposis colon cance Werners syndrome Xeroderma pigmentosum Frequency

1/22,000 in some popns. 1/200 3/1,000,000 1/250,000 Defect Deficient excision repair

Deficient mismatch repair Deficient helicase Deficient excision repair DNA Repair DNA Damage Can Activate Expression of Whole Sets of Genes

Heat Shock Response SOS Response DNA Repair 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 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.

DNA DAMAGE SURVEILLANCE RECOGNITION SIGNALLING REPAIR

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