weber uiuc edu 28 September 2007 MCB150 Lecture 15 Lecture 15 28 September 2007 Announcements More broken hand notes The audio for this lecture was donated by a student and is of poor quality so these notes should be closely inspected for errors Hurdles to Overcome DNA POLYMERASE ONLY WORKS IN THE 5 TO 3 DIRECTION o That is the parental strand is read in the 3 5 direction but the daughter strand is synthesized on the 5 3 direction DIAGRAM o Be able to diagram the direction of synthesis The bottom strand of the upper left will add in the correct direction Nothing special there If we need to move in both directions then the right side has problems It CANNOT add nucleotides from 3 to 5 CANNOT DO THIS N A synthesis occurs in the 5 to 3 direction ONLY The problem is how do we move away from the ori in the 3 to 5 direction DNA Synthesis The 5 to 3 synthesis strand is handled normally o This is called leading strand synthesis o It will keep going until the end The 3 to 5 synthesis will happen in piecemeal because it has to proceed in 5 to 3 o This is called lagging strand synthesis o This will be made in small pieces in the 5 to 3 direction o The fragments are called Okazaki fragments The gumdrop example recall his work up on the stage o Know that you can t go from 3 to 5 o Need to jump ahead then work backwards Lagging strand synthesis will move over to where helicase is working then move back to the ori o Go as far forward as you can then work backwards o Every Okazaki fragment ends when it runs into the previous one or the ori area which has already been synthesized by the leading strand o Repeatedly do this Synthesis Diagram Helicase spreads and single stranded binding proteins keep it open As soon as DNA is unwound the leading strand synthesis begins o Can do this on both parental strands just in opposite directions o This is because the second strand is anti parallel As soon as the DNA is unwound enough then the first Okazaki fragment starts o Okazaki fragments are built on both strands too o The first fragment works back to the ori where it runs into the leading strand Wait until more DNA has been opened further Page 1 of 4 weber uiuc edu 28 September 2007 MCB150 Lecture 15 o Another fragment starts up and works backwards to the first Okazaki fragment o This keeps repeating the entire time until the DNA is done The whole time the leading strand continues unabated continuous synthesis The lagging strand is discontinous DNA synthesis This is how it happens Problem 2 The DNA Polymerases We are still using E coli in this discussion DNA polymerases make DNA from nucleic acids They CANNOT start DNA strands from scratch o Even if they know what is supposed to go there o Can ONLY link to a free 3 hydroxyl OH group Primase is an RNA synthesizing enzyme o It will hybridize the DNA by adding RNA o Terminology DNA dependent RNA synthesizing enzyme You use the DNA as the template RNA is what is created o But why use RNA Because RNA must be added in to give a primer to which DNA polymerase can attach to the 3 hydroxyl It will add RNA bases and create covalent linkages between them It lays in the base pair then performs nuclei acid synthesis by following the 5 3 synthesis Will add in uracil when needed o The requirement is a free 3 hydroxyl In theory it could do its job by adding in just one base pair However the bases are only hydrogen bound and if it is an AU pair then it isn t very strong So it adds in a few more o Once at least one is added DNA polymerase has what it needs DNA polymerase III has what it needs and stars on its trip Priming the Pump Pay close attention to the orientation of the parental strand o THIS WILL BE ON THE EXAM KNOW THE DIRECTIONALITY There is no magical first base to start Primase will recognize the base and insert the correct RNA base o No hydrolysis of the phosphate was necessary because it is only hydrogen bonds that are forming Now there is a free 3 hydroxyl group Another base is added on to the strand via hydrogen bonds Now every NTP coming in will have its phosphate hydrolyzed and will be linked into the chain to the 3 hydroxyl group in the chain Primase only knows NTPs which are rNTPs Now DNA polymerase III pol III comes in and rips away It is a very processive enzyme efficient and rapid If it is the leading strand it will keep going until told to stop QUESTION as soon as helicase has done its job primase can move in It is more about space than the number of bases per se Primase will begin as soon as it can It is a timing issue Page 2 of 4 weber uiuc edu 28 September 2007 MCB150 Lecture 15 Every single new strand has to be primed including all of the Okazaki strands o Every new piece of DNA has to be primed DNA Priming Diagram Primase starts at the ori then lays down a few RNA bases After a few bases primase is displaced from the replication fork by DNA polymerase III DNA pol III zips along until it either finds the terminator sequence or it runs into the start of an Okazaki fragment or runs off the end in eukaryotes Leading vs Lagging Synthesis Leading strand synthesis occurs as fast as helicase can unwind the DNA Once unwound far enough primase moves in and primes with RNA in the lagging strand Then DNA pol III gets in and adds DNA in the lagging strand Synthesis in lagging starts away from the ori then moves back towards it This continues until it runs into a primer Cleanup of RNA Primers Need to remove the RNA primers Do not want RNA litter in the DNA Otherwise over time you will have a hybrid DNA RNA genome There is also a nick in the backbone where the Okazaki fragments are not yet joined covalently o Gaps are when there are bases missing o Nicks are when there is a discontinuous phosphodiester backbone Every Okazaki fragment is its own nucleic acid with its own 5 and 3 end There are cells that can get in at a nick and remove the RNA bases DNA polymerase I will pluck out the RNA and replace it with a correct dNTP o rUTP dTTP uGTP dGTP etc DNA polymerases require the 3 free hydroxyl This is ok because there is a 3 available at the end of the previous fragment You will end with a nick Why Because the removal of an RNA will leave a gap o DNA polymerase I cannot ligate that nick because there isn t a nucleotide being added …