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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 Presentation

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BS2009 - GENOMESDNA 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

S phaseDNA synthesisG2Growth phase 2MMitosis G1Growth phase 1GoQuiescentMain checkpointsTHE EUKARYOTIC CELL CYCLE REPLICATION GENERAL PRINCIPLES

STARTMust be ready: G1Must 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) REPLICATION GENERAL PRINCIPLES

ACCURACY/FIDELITYProof reading/repair (G2)Must distinguish between original and copy (EPIGENETICS)

SummaryDNA ReplicationOccurs during S phase of the cell cycleSemi-conservative (Meselson & Stahl)5 3directionleading strandlagging strand (discontinuous)RNA primerSummarySingle origin in bacteriaMultiple origins in eukaryotesBi-directionalARS elements from yeastcloned origins

SEMI CONSERVATIVE535OH 3535535OH 3OH 3DNA polymeraseDNA synthesis always occurs by addingnucleotides to the 3-OH of the growing strand.

Synthesis is always in the 5- 3 direction

E. Coli: DnaGSSBDnaB

DnaB

(SSB)Replication EnzymesDNA Polymerase - Matches the correct nucleotides then joins adjacent nucleotides to each otherPrimase - Provides an RNA primer to start polymerizationLigase - Joins adjacent DNA strands together (fixes nicks)Helicase - Unwinds the DNA and melts itSingle Strand Binding Proteins - Keep the DNA single stranded after it has been melted by helicaseGyrase - A topisomerase that Relieves torsional strain in the DNA moleculeTelomerase - Finishes off the ends of DNA strands

exonuclease

Eukaryotes vs ProkaryotesEnzymology, fundamental features, replication fork geometry, and use of multiprotein machinery conservedMore protein components in Euk replication machineryReplication must proceed through nucleosomes Replication fork moves 10X faster in Prok. REPLICATION GENERAL PRINCIPLES

START

FINISH

ACCURACY/FIDELITY

Accuracy and Fidelity

Maintenance of DNA SequencesMaintenance of DNA SequencesDNA Polymerase as Self Correcting EnzymeCorrect nucleotide has greater affinity for moving polymerase than incorrect nucleotideExonucleolytic proofreading of DNA polymeraseDNA molecules w/ mismatched 3 OH end are not effective templates; polymerase cannot extend when 3 OH is not base pairedDNA polymerase has separate catalytic site that removes unpaired residues at terminusMaintenance of DNA SequencesDNA Polymerase as Self Correcting EnzymeTwo catalytic sites

Strand Directed Mismatch Repair System

Removes replication errors not recognized by replication machineDetects distortion in DNA helixDistinguishes newly replicated strand from parental strand by methylation of A residues in GATC in bacteriaMethylation occurs shortly after replication occursReduces error rate 100X3 Step Processrecognition of mismatchexcision of segment of DNA containing mismatchresynthesis of excised fragment

Strand Directed Mismatch Repair in Mammals

Newly synthesized strand is preferentially nicked and can be distinguish in this manner from parental strandDefective copy of mismatch repair gene predisposed to cancerStrand Directed Mismatch Repair System

Causes of DNA DamageChemical mutagensRadiationFree radicalsRADIATION = ENERGY

ENERGY DEPOSITION IN DNA

DNA DAMAGE33

34 S-A.T-SP P S-A.T-SP P S-T A-SP P S-GC-SP P S-C.....G-SP P S-T..A-SP P S-A..T-SP PP 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 WATERH2O OH + H + e-..OHOH..35Carbohydrate + O2 Energy + CO2 + H2O

RESPIRATION AND AGEING02 H2O2 OH..DNA damage~10000 lesions/cell/day36DNA RepairTypes of DNA Damage: Base Loss and Base Modification

Depurination

Chemical ModificationPhotodamage thymine dimerChemical Modification by O2 free radicalsDeaminationDNA Repair

Despite 1000s of alterations that occur in DNA each day, few are retained as mutationsEfficient repair mechanismsImportance of DNA repair highlighted by: Number of genes devoted to DNA repair mutation rates with inactivation or loss of DNA repair geneDefects in DNA repair associated with several disease states38DNA replication and repair disordersDisorderFrequencyDefectFanconis anaemia1/22,000 in some popns.Deficient excision repairHereditary nonpolyposis colon cance1/200Deficient mismatch repairWerners syndrome3/1,000,000Deficient helicaseXeroderma pigmentosum1/250,000Deficient excision repair39DNA RepairDNA Damage Can Activate Expression of Whole Sets of GenesHeat Shock ResponseSOS Response

DNA Repair

DNA Repair

Base Excision RepairDNA glycosylase recognizes damaged baseRemoves base leaving deoxyribose sugarAP endonuclease cuts phosphodiester backboneDNA polymerase replaces missing nucleotideDNA ligase seals nickFailure of DNA repairWhen 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.44

DNA DAMAGE

SURVEILLANCE

RECOGNITION

SIGNALLING

REPAIR46