BSCI222 Lecture 3 9 10 13 So DNA is the genetic material How does it function What are its functions o Needs to make copies of itself Needs to code for amino acids generally encode information Has to be able to mutate if always perfect no evolution Has to be complex Has to express the information that it carries transcription Carry info replicate express transcribe modify mutate Once the model came out started theorizing about DNA replication o 3 major models Conservative 2 original strands stay together copy made Semiconservative strands separated each of the 2 copies made with 1 old and 1 new or Dispersive bits of each end up in both of the 2 final copies o Technology Meselson and Stahl Cesium chloride density gradient centrifugation Very fast centrifuge with very heavy salt spin and the cesium starts getting pulled down while also diffusing back up to settle the gradient So one side of the tube will be heavier than the other Very sensitive way to measure differences in densities The longer DNA gets centrifuged the more it ends up in the middle neutral buoyancy takes about 36 hours of spinning Can grow E coli in heavy or normal Nitrogen 14 or 15 thereby changing the density of the DNA The stuff grown in N 15 will end up deeper in the tube heavier Analytical centrifuge 2 Heavy strands put in N 14 medium anything new will be light After 1 generation expect 2 bands of DNA each with one heavy and one light strand each full band is N 14 5 After two generations expect 4 total DNA bands 2 N 14 5 and 2 N 14 All DNA replication is semi conservative One parent strand and one newly synthesized daughter strand Minimal requirements for DNA replication o 1 Template of single stranded DNA o 2 Deoxyribonucleotide triphosphates dNTPs o 3 DNA polymerase enzyme o 4 A short oligonucleotide primer RNA or DNA o 5 A free 3 OH group to extend keep adding nucleotides The 3 OH group of the last nucleotide on the strand electrons from the Oxygen to the Phosphorus attacks the 5 phosphate group of the incoming dNTP diphosphate gave the energy to drive the reaction 2 phosphates are cleaved off A phosphodiester bond forms between the 2 nucleotides and phosphate ions are released DNA is always synthesized 5 to 3 Start replication at the end of a DNA molecule split the helix apart One strand will have a 5 end the other strand will have a 3 end Synthesis goes right to left on one strand having to start somewhat in the molecule and work back out toward the end called the lagging strand synthesized in a series of fragments that can t start until the helix is opened up a little further and left to right on the other strand called the leading strand o Leading strand has continuous synthesis lagging strand has discontinuous DNA synthesis fragments called Okazaki fragments o Helicase opens the helix breaking off hydrogen bonds RNA polymerase will make the first primers DNA polymerase will grab on in order to make sure the hydrogen bonds don t come back together starts replicating from template The lagging strand is different after helicase has gotten pretty far in a different RNA polymerase will make a short RNA sequence on the DNA template which will be the handle on which the DNA polymerase can bind and start DNA synthesis Once the handle is there the DNA polymerase can t get rid of the last RNA sequence that was there so second polymerase must come and chew away the RNA leaving a gap between the Okazaki fragments Then need DNA ligase to join the two Okazaki fragments together o Have to open up a circle somewhere in the middle of the DNA molecule TEST diagram the replication fork Separate the 2 original strands there and can do continuous synthesis on both in the 5 to 3 directions Anything that is a lagging strand relative to where the origin point is has to be done the fragmented way Takes a group of proteins to open the helix initiator proteins recognize the oriC origin of replication bind to it causing a short stretch of DNA to unwind allowing the helicase and other single strand binding proteins to attach to the single stranded DNA Normal relaxed DNA strands about 10 bases per turn Add extra turn extra twist increase in linking number Can also decrease the linking number DNA is normally negatively supercoiled a little underwound meaning it s a little more accessible to proteins When these proteins bind they re inducing a little bit of untwisting in order to open up a spot in the helix to let the next enzymes in The next enzyme is helicase opening it up spreading the ends jamming the twists up at the top supercoiling making it harder and harder to proceed until DNA gyrase relieves the strain ahead of the replication fork DNA gyrase topoisomerase enzyme that can turn the topology of the DNA There are Type I and Type II topoisomerases depending on whether they depend on single nick on the strand or double stranded break DNA gyrase is Type II always changing the number of twists linking number by 2 Open the bubble continuing the opening with helicase gyrase relieving the strain Primase molecule comes in makes a short piece of complementary RNA to the DNA to make the first bit of double stranded nucleic acid in this region After that DNA polymerase III can come in which does most of the DNA synthesis all the continuous on the leading strand all the red fragments on the lagging strand DNA P III does not have a 5 to 3 exonuclease activity has a polymerase activity can synthesize but when it runs into something else on the template like the RNA primer laid down by the Primase doesn t have the exonuclease activity to get rid of the primer Has to stop DNA Polymerase I which is different and DOES have 5 to 3 exonuclease activity As it chews up the primer 10 20 bases re makes the DNA template Why not just use DNA P I for everything It s slower and more error prone So P III is better for doing 80 of the work and just use P I for clean up Okazaki fragment RNA primer empty space where P III is working right to left 3 to 5 original RNA primer where P III started Once P III reaches that second RNA primer stops and P I has to move it and then finally DNA ligase will join the old fragment and the new fragment together Replication forks are found in many different configurations of replication o Theta replication of circular genomes single origin of replication halfway through see a structure that looks like the Greek letter theta All the steps are there initiator proteins stabilize the bubble etc Follow it all the way around