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Lecture 4

BIO240H Lecture 4.doc

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Jennifer Harris

Lecture 4: DNA Replication (part II) Lecture Outline: • Issues in replication • DNA repair Readings: Alberts textbook, Ch 5, pp. 276-288, 292-304 Monitoring yeast replication - Showing how fast DNA replication goes. - Micro-rays: in each location on micro-ray, certain DNA sequence has been spotted & it sticks there. - Have some yeast, then add fluorescent dNTPs, stimulate DNA replication, isolate DNA & apply it to micro-ray & it will hybridize to specific sequences on micro-ray. - At 20 minutes, DNA is fully replicated. - Do experiment where you synchronize population of yeast cells in that you arrest them right before replication begins & then stop & harvest them at certain time points after allowing replication to continue – can see how replication actually progresses in yeast genome. After you harvest each 1 of these populations of yeast that you allowed to replicate for different amounts of time, separate strands & fluorescently label them & then you hybridize them to what is know as micro-ray. - In cases where you do not have any replication (kind of like control – 0 min) – see light green dots indicate unreplicated DNA while replicated DNA are indicated by dark green dots. As you progress, have larger patches of DNA – micro-ray itself is composed of yeast genome in little fragments that are arranged sequentially from left to right. - This experiment tells us replication in fact begins at origin of replication & moves by bidirectionally out from that origin. - In real yeast genome, would have hundreds of origins all that are moving out from origin. Issues in DNA Replication 1) What happens at ends of chromosomes? 2) How is DNA unwound? 3) How are mistakes found & corrected? What happens at ends of chromosomes? - What happens when fork reaches end? Problem at ends of chromosomes Leading strand: Why is shortening of 5’ end of daughter DNA potential problem? Loss of info Lagging strand: For which strand is this problem? Lagging - Lagging strand: it needs enough space to be able to synthesize primer so that DNA polymerase can replicate template but at end of 3’ end of lagging strand, it doesn’t have enough room to do this. - This means in every cycle of replication, if this issue is not dealt with, then you would have potentially loss of info contained at 3’ end of lagging strand. In each round daughter cell that got lagging strand template would actually have less DNA than parental cell. What happens at ends? In majority of eukaryotes, this problem is solved by having repetitive DNA sequences at ends of chromosomes. Ends are called: telomeres - DNA sequence that don’t contain any info in it – place-holder so that replication of real DNA (important stuff) is maintained without any loss. Telomerase to rescue Repetitive sequence that is added to 3’ end of parental strand (i.e. lagging strand template) is determined by RNA in telomerase. RNA  DNA copy - Has contained within enzyme itself an RNA template that it uses to guide addition of new sequences onto 3’ end – elongating 3’ end of strand using DNA. Synthesizing RNA-DNA hybrid. RNA template contained in telomerase can vary depending on species – tends to be fairly similar & fairly rich in C-G’s – generates tandem repeats based on this template. Telomere Replication Telomerase: 1) RNA template 2) Resembles: reverse transcriptase 3) Prefers G-C rich ends 4) Adds nucleotides to 3’ ends - Have incomplete newly synthesized lagging strand, not enough space for DNA replication to happen on lagging strand – telomerase comes along with RNA template in it – binds to 3’ end of parental strand & adds additional nucleotides based on template in consecutive steps. Can add many copies of these repeats in tandem with each other (doesn’t add just 1) - Using RNA template but synthesizing DNA strand makes it resemble enzyme called reverse transcriptase (do not have inherent RNA template – can use any strand of RNA as its template). - What it’s doing is generating lots of ssDNA (vulnerable to forming kinks, H-bonds, etc) – in fact structure of most telomeres is such that it folding back on itself is good thing b/c it protects telomeres from degradation. Another reason why G-C bonds at 3’ ends is good thing – form very stable hairpin loops that protect DNA from further degradation. Telomere length - Found to be tightly regulated processes in organisms – tied to # of cell divisions or replication. - How do we know telomere length is tightly regulated? Experiment: artificially elongate chromosomal end by adding on more copies of telomeres & put back into yeast cells & as # of cell divisions progresses, assay how long telomere repeats are. - As # of cell divisions progresses, cells decrease/increase length of telomere back to what it should be. - In most cells in body, there is limited # of cell divisions which are optimal & our somatic cells are actually born with specific # of repeats. As cell divisions progress, lose certain # of nucleotides with every division if telomerase is turned off. Some cells actually use this as way of halting replication after certain # of cell divisions so eventually enough of chromosome is lost that cell stops dividing – replicative cell senescence – important safeguard against uncontrolled proliferation that if left unchecked could lead to cancer. “Winding problem” 1) Unwinding DNA is energetically: expensive 2) Supercoils in same direction from twist of double helix: + 3) Opposite direction: – 4) Replication introduces supercoils in which direction? + - Positive supercoil: supercoil is coiled in same way single strands are twisted. Negative supercoils are twisted in opposite way. When you have positive supercoils, make it harder to separate DNA. When you introduce negative supercoils, allows them to separate out. - Have to relive tension in strands – relieve supercoils. - When you’re unwinding 2 strands, you’re doing so in context of gigantic chromosome – this means that in order to unwind things, ends of chromosomes have to rotate & rotating huge chromosome is energetically incredibly expensive & slow. As you pull strands apart, in direction of replication those coils are getting tighter – supercoils in same direction of twist of double helix – as you pull them apart in direction of replication are positive. In opposite direction is negative coils. Topoisomerase 1) Type of break: single-stranded 2) This allows DNA to: rotate around backbone. - Type 1 makes single-strand break & this single strand is twisted (swivels) around each other – relieves tension in bond. - Attacks phosphodiester backbone of single strand & breaks it only on 1 side – only 1 side is necessary – this is to alleviate supercoils in order that it can rotate around & not be so twisted. Breaks only 1 strand allowing for this rotation to happen so DNA can rotate around its backbone. Once this happens, it dissociates & you have spontaneous re-formation of phosphodiester bond & regenerates DNA helix. - Most effective at alleviating positive coils just ahead of replication fork. Topoisomerase II 1. Type of break: double stranded 2. This allows: helices pass through one another (either making/breaking supercoils) - Breaks double helix - Again this is done b/c DNA as it unwinds can be tangled very quickly. - Deals with what happens when you get 2 strands of DNA that
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