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Lecture 10

Lecture 10 - Reproduction - BIOL308.pdf

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University of Waterloo
BIOL 308
Bruce Wolff

Lecture 10Intro to DNA replicationSpecific proteins recognize specific sites on NA and bindDNA RNA protein interactions very importantEnzymaticnuclei acids substrate protein enzymesDNA restriction enzymes topoisomerases telomerases methylases polymerasesRNA tRNA synthetases polymerases polyadenylasesStructural change in DNARNA structureHelicases unwinding parental DNA duplex during DNA replication histones TBP etcRegulatoryIn both transcription DNA binding and tranlation RNA bindingTypes of protein NA interactions charge to chargeHydrogen bondsVan der waals interactionHydrophobic interactionsKeep In MindDNA and RNA are negatively chargedBinding proteins tend to be positively chargeed ie HistoneDNA and RNA are surrounded by positively charged ionsWhen protein binds to nucleic acid positively charged ions are displaced energy requirement etcMajor and minor grooveMajor is more involved in binding because it is more specificDNA replication Initiation of ReplicationLearning objectivesSemiconservative and bidirectionalDescribe the cis elements and trans factors involved in the process of replication initiationDescribe how ori sequences are located and studiedDNA ReplicationReplication is semiconservativeEfficient processit always starts at the defined sequence of base pairsReplication origin a stretch of DNA necessary and sufficient for beginning of replicationBacterial and plasmid genomes often have only one more complicated in eukaryotic cell multiple originsEach region served by one DNA origin in repliconAny one piece of DNA which replicates as a single unitBidirectionalfrom the origin in majority of oranisms2 replication forksReplication bubbleLeading and lagging strandBidirectional ConfirmationCultured mamalian cellsHigh and low concentrations of HTDNA labeled near replication originDNA dried on a microscope slideAutoradiographic patternsThe spots are not labeled because of RNA primersHow were the enzymes involved in DNA replication discoveredDNA replication first studied in prokaryotesusing both genetic and biochemical approachesMake a mutant populationand screen for mutants affected in DNA replicationCharacterizeas to whether replication stops quickly or slowly under nonpermissive conditionsPurify enzymesrequired for replication in vitroAssayadd radiochemical dNTP to an extract containing enzymes necessary for replication along with a template DNA and measure incorporation of radioactivity into new DNA Can be precipitated and collected on a filter NOTES FROM LECTURES Page 1
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