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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 pulleddown, 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. After1 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 DNAo 2. Deoxyribonucleotide triphosphates (dNTPs)o 3. DNA polymerase enzymeo 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 bethe 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 theoriC (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 TypeII 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 exonucleaseactivity 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. -


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UMD BSCI 222 - Lecture 3

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