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University of Toronto Mississauga
Gordon Anderson

DNA replication (Part 1) - 3 activities of genes genes carries information - Genetic material needs to be passed unchanged from generation to generation General Features Major Proteins 3 stages General Features Semi-conservative 1 parental strand paired with 1 daughter strand Semi-discontinous Unidirectional or bidirectional Semiconservative; Dispersive (mixture of parental and daughter strand) Fig 20.1: Conservative 2 parental strands paired together and 2 daughter strands paired together Fig. 20.2- Evidence of Semi-conservative: CsCl gradients - Took bacteria cells and grew in medium containing light form of nitrogen (N14); dark form of nitrogen (N15). - Centrifuge on CsCl gradient in tube; N14 and N15 DNA moves down and stop when it matches density of CsCl; add ethidum bromide and binds to DNA so can see heavy and light bands under UV light ; compare 2 intensities (heavy/ light bands) Fig.20.4 : after 2 replication: heavy bands disappearing and see light and medium bands appearing Fig.20.3: Meselson and Stahl (review) Rifampicin inhibits DNA replication Rifampicin inhibits RNA polymerase (not DNA polymerase) Fig. 20.7 Semi-discontinous- DNA polymerase synthesizes 5 to 3; DNA polymerase needs a primer (RNA or DNA); Fig.20.5: Leading and Lagging Strands Continous, Semicontinous, discontinous synthesis must occur in opposite directions (one strand continous and one strand synthesized in short fragments called Okazaki fragments) Fig 20.6: Okazaki fragments -using E.coli, replicate T4 DNA - Radioactive thymadine added so can label DNA synthesized. Pulses from 2- 120s. - At the shortest times, have many short DNA pieces. - As time passes, see longer pieces of DNA being labelled. - Therefore, newly formed short DNA pieces (Okazaki fragments) being attached to pre-formed larger DNA pieces (dont see larger DNA because formed before labelling) Therefore, short pieces of Okazaki being produced Fig 20.7- Priming in DNA synthesis: Replication Fork - point at which DNA separates to allow replication; RNA primers used Fig 20.8- Finding and Measuring RNA primers (10-12 nt): Okazaki fragments not completely destroyed when treated with DNase. - Rifampicin- inhibits DNA replication; inhibits RNA polymerase - If RNaseH and polymerase I absent from mutant cells, then have good yield of primers (10-12nt) and intense band produced Fig 20.9 Theta Mode of Replication - One whole generation of DNA replication occurred in presence of radioactive label - 2 replication forks (X and Y); cant tell from unigraph if have unidirectional or bidirectional. - Next experiment to distinguish between two types of replication Fig 20.10 Bacillus subtilis and E-coli Experiment showing Bidirectional DNA replication - Low level of radioactivity pulse given initially, then cells transferred to high level of radioactivity pulse; (p.646) - DNA is being labelled at both ends w/ higher isotope ->synthesis occurring at both ends Fig 20.11 Drosophilia melanogaster and Amphibian cells - Bidirectional ; high dose of radioactivity given first and then lower dose Eukaryotes have many replicons while prokaryotes have only one replicon Fig 20.12 Plasmid colE1- Unidirectional (p.649) - cut circular plasmid with EcoR1, provide marker, linearize plasmid DNA, watch replication bubble- bigger downwards As replication proceeds, bubble gets bigger and moving downwards while part above bubble remains the same. Therefore, have unidirectional replication Fig 20.13 Rolling Circle Mode (ssDNA) phage (circular DNA phage) 174 - negative strand acts as template strand - addition of dNTPs to 3 of positive strand and positive strand displaced and cleaved. - Then forms more positive strand DNAs for packaging (positive strand same sense as mRNA) Fig 20.14 Bacteriophage (dsDNA) - phage has ds linear DNA (inject linear dsDNA into bacterial cells) - cos overhangs complementary so can anneal and circularize; circularized DNA as template DNA - Initially, phage replication occurs by theta mode of replication to produce several copies of circular DNA (acts as template for rolling circle replication after) - The displaced strand is now template for lagging strand synthesis (different from X174) - concatemers produced genome length of DNA (cleaved at cos sites), packaged into heads DNA Replication (Part 2) Enzymes Required for DNA Replication Helicase- Separates 2 DNA strands SSB (single stranded binding proteins) - Keep strands separated; prevents reannealing Topoisomerase prevents winding of DNA DNA polymerases synthesizes Fig 20.21 DNaB is the Helicase DNaG and ssB enhance separation DNaB- cause of separation, helicase activity - Pairing a large circular DNA from M13 annealed to a complementary short fragment labelled. As long as large circular DNA and short DNA annealed, both show up - If separated, long fragments doesnt show up, only small fragments - In presence of DNaB, see separation and see small fragments - Adding DNaG and ssB to DNaB enhances helicase activity - Therefore DNaB is the cause of the separation and has helicase activity - ssB proteins prevents reannealing of separated strands and stabilize them work with cooperative binding- once one attaches, stimulates binding of other ssB proteins so have chains of ssB proteins being attached - ssB proteins also stimulate their specific DNA polymerases required to replicated DNA (ex E-coli ssB proteins stimulate E-coli polymerases) Stimulation is specific; Therefore, ssB proteins essential for replication - ssB bind DNA proteins in non-sequence specific manner As one strand unwound, get more winding on other side 2 types of supercoiling:
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