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Lecture 15

BIO240 Lecture 15

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University of Toronto St. George
Jennifer Harris

Monday, September 15, 2008 Lecture 3: Intro to DNA Replication Lecture Outline: 1) Overview of DNA replication 2) DNA replication in bacteria Readings: Alberts textbook, Ch 5, pp. 263-276, 281-287 - Only time you need DNA replication is when cell divides. - Every single cell in organism’s body contains entire genome required to build entire organism. Questions of day 1) Is DNA replication conservative or semiconservative? 2) What is direction of DNA replication? 3) Where does DNA replication start? - Watson & Crick worked out structure of DNA – used x-ray crystallography & models. Is DNA replication conservative or semiconservative? 2 hypotheses: 1) DNA synthesis is conservative 2) DNA synthesis is semiconservative Red strands? – Parental Blue strands? – Newly synthesized - Conservative – 1 daughter cell (when cell divides) gets all of original parental strand & other gets completely new synthesized strand. - Semiconservative – use both strands as template, 1 of original strand goes into 1 cell & other goes into other cell. This hypothesis is right. Both daughter cells get 1 of original strand & 1 new synthesized strand. - Important implications for error rates – if you have 1 original copy & 1 newly synthesized copy, important implications for how error checking may happen, how mistakes might be corrected, etc. What is direction of DNA replication? 3 possible models: 1) Unidirectional growth of single strands from 2 starting points. Ex: linear virus 2) Unidirectional growth of 2 strands from 1 starting point. Ex: some plasmids 3) Bidirectional growth from 1 starting point. – This is most common type of replication. Ex: eukaryotes Where does DNA replication start? 2 possibilities: 1) Always start from same location on DNA – have some control over process – some consensus sequences that are recognized as initiation for replication. What are some of characteristics of sequences at replication origins? st - Easy to open, A-T rich – 1 step in initiating DNA replication is to tear 2 strands of DNA apart b/c you need to use 1 of them as template to synthesize new strand. A-T base pairs are weaker than G-C base pairs – 2 H-bonds rather than 3. - Attractive to initiator proteins – bind to specific sequences – proteins that help assemble machinery of DNA replication at that site so that replication can begin. 2) Random start How many origins of replication? 1) Single – 2) Multiple – Eukaryotes Bacteria - Ex: Yeast chromosome #3: 17 chromosomes & 400 origins interspersed at regular intervals – generally spaced at about 30 kilobases apart – can be replicated in about 8 minutes. If you start deleting some of these origins, replication machinery is fast enough that it can compensate for those losses but once you get to certain # of losses, it actually overwhelms system & start to lose ends of chromosomes. How do you identify origins of replication? What does ARS stand for? Autonomously replicating sequences - Plasmids – found normally in bacteria – can be inserted in any piece of DNA. - Purpose of experiment: what pieces of yeast DNA is the origin of replication? - Randomly chopped up random pieces of chromosomes & insert it into thousands of plasmids. Take those plasmids & insert them into yeast cells – 1 yeast cell gets 1 plasmid. Is there a sequence that will direct DNA to be replicated? Origin of replication is named ARS. Grow them on medium – need to find way to select yeast cells that contain origin of replication. Yeast with its DNA machinery will make many copies of plasmid. - Left: no replication of plasmids – no ARS – contains very few plasmids. - Also include on plasmid gene called His gene – allows bacterium (yeast) to grow in medium without AA histidine. - Take entire yeast genome & chop it up in little pieces – put little pieces into plasmid vector. This plasmid vector has gene which synthesizes histidine – significance of this ARS sequence is that it is term defined by this assay as autonomously replicating sequence which means that if it contains this sequence this plasmid will be able to replicate itself. - If you have whole bunch of plasmids, each with slightly different portion of yeast genome, some of those plasmids will have ARS sequences. If you introduce all of plasmids into yeast cells that actually lack this His gene, that means that these yeast cells cannot actually grow in absence of histidine & then you played it on selective medium without histidine, what you will find is that if that particular yeast colony has ARS sequence in it, it will be able to replicate & therefore replicate His gene & therefore grow in medium without His & then you can take these yeast colonies, extract plasmid from it, sequence piece of yeast genome & determine what the sequence which is putative origin of replication might be is. - ARS sequence is not entirely synonymous with origin of replication, it is putative origin of replication as defined by this assay but in fact when you look at those sequences that result in ability to grow in medium with His, many of them turn out to be origins of replication. - In rare cases, plasmid without ARS sequence actually integrates into host genome & gets replicated there – very rare event but these colonies are result of that – false positives. How does DNA replication proceed in bacteria? This style of replication only applies to: circular genomes - Have single origin of replication. - When replication begins, it proceeds bidirectionally – there are actually 2 replication forks & eventually those 2 forks meet & you get dissociation & you have 2 complete daughter DNA molecules. - 1 of simplest systems. What happens at DNA replication forks? - In replication fork, there is 2 mechanisms/ways to replicate DNA: leading strand (copy of parental) & lagging strand. Proceeds to right – bit choppy for lagging strand. - Elongation of DNA strands in newly synthesized DNA always happens in 5’ to 3’ direction. At replication fork, have leading strand (there is an asymmetry that is happening here) that is able to be synthesized continuously as replication fork moves in this direction & strands separate whereas lagging strand is not able to synthesize DNA continuously – got to go in ratcheting fashion (backwards) – has to be synthesized in short chunks. Overview of DNA replication Procedure: 1) Separate DNA strands 2) Synthesize DNA 3) Proofread newly synthesized DNA (copy has to be exquisitely accurate) Ingredients for synthesis: 1) Origin (certain sequence of DNA that initiator can recognize) 2) Primers (DNA polymerase can only elongate from existing primer sequence) 3) dNTPs (deoxyribonucleoside triphosphates – building blocks of nucleic acids) 4) ATP (for energy) 5) DNA polymerase (major enzyme that forms nucleic acid chain) 6) Accessory proteins DNA synthesis Base pairing: A-T, C-G - Actual process of DNA synthesis is accomplished by polymerase. Incoming nucleotide is determined by necessary base-pairing interaction with next AA in template strand. - Elongating chain is going from 5’ to 3’ – template strand is in opposite direction. - Requirement is for DNA polymerase to keep elongating is that you have correctly base-paired
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