are doing a synthesis reaction. Which can be a sequence of DNA primer? Must go from 5’ to 3’ Its reading DNA from 3 to 5 Summary of DNA Replication Starts at ori Helicase unwinds (ATP) SSB proteins keep strands apart Primase makes RNA primer DNA polymerase III adds nucleotides to 3’ OH of RNA primer Strand grows in 5’ to 3’ direction We have to have a different mechanism for making the second strand of DNA because DNA polymerase III can only go from 5’ to 3’. Need second mode of synthesis. We just described leading strand synthesis 5 to 3 , other strand is going to be a sequence of very small molecules that are going to be added together to synthesis the opposite strand called lagging. A series of 5 to 3 prime chunks that are then going to have to be put back together later. Each piece is called an okazaki fragment, so we must put each piece together to make a complete strand. Need rna primer for each fragment.
Okazaki Fragments • Structure is short chain of RNA at 5´ end followed by a run of DNA • Must get rid of RNA – DNA Polymerase I is a repair enzyme – Has 5´ to 3´ exonuclease activity – Therefore, can “chew” off RNA primer, use preceding Okazaki fragment as new primer, and replace RNA with DNA CQ: is this leading or lagging strand synthesis Lagging since, the 3’ is first okazaki fragments But we must get rid of RNA , so DNA polymerase I comes in and cuts out all the RNA pieces and replaces them with DNA. Eventually we have a bunch of fragments we must connect them back together final enzyme comes in called DNA ligase puts the knits back together So describe leading and lagging synthesis: Helicase unwinds, Leading: The 3’ end exposed can have leading synthesis. We can start our RNA primer 5’ end added to the 3’ end. Works fine. Lagging: The other strand has the 5’ end exposed so we can just start building, so hopes up the DNA strand and adds a primer and then the makes okasaki fragment, then we need to get rid of primers, then use ligase to put the fragment together.
CQ: Step put in order: Helicase unwinds DNA Single stranded DNA is bound by SSB proteins A small piece of RNA is synthesized by primase A new DNA is strand is synthesized by DNA pol III RNA primer cleave out New DNA is synthesized by DNA Pol I to replace primer Ligase seals gap in okasaki strand Finishing up Replication: Sliding DNA Clamp One more enzyme of importance: the DNA clamp, is what binds DNA polymerase III to the strand that is being replicated. Since DNA polymerase moves so fast and could disengage this enyme makes sure the DNA polymerase III stays connected to the strand that is being replicated. Without it we would just have fragments of DNA that are not being put together. Telomeres; the ends of the DNA were the RNA primer is being laid down. The 3’ end. And everytime they come to replace that RNA template to put in new DNA it actually shoots off the end of the fragment. So you lose a little part of the DNA everytime you replicate it. That’s a problem. DNA continues to get shorter and shorter. So there is an enzyme called telomerase which sits there and goes and puts back on those end to the DNA to ensure that you are not losing DNA every time you replicate it.
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