Bio Final: Nov 26 DNA Replication  “Tool Kit” Begins at Ori C (origin of replication)o Bacteria have one Ori C because they have a singular circular chromosome When DNA is unzipped (breaking hydrogen bonds), we have two replication forks that proceed in opposite directions and meet on the opposite side of the chromosome, then the chromosome is then replicatedo Eukaryotes have 46 chromosomes and each chromosome has many Ori C’s Enzymes1. Helicase is the enzyme that unzips the DNA molecule by breaking the hydrogen bonds that are holding the bases togethera. “Tab on blue jean zippers”2. Gyrase the enzyme that relieves supercoils up stream of the helicasea. “Telephone cord”3. DNA Polymerase I the idea that is can create a polymer of nucleotides a. Very large complex enzymeb. Functions SLOWLYc. Has three activities functioning as:i. 5 1  3 1 Polymerase Ability  DNA Molecule is structurally polarized and the two polynucleotides are antiparallel (strands are oriented in opposite directions) Adds Nucleotides to the 31 end of other nucleotides Allows the addition of new nucleotides to the 31 hydroxyl end of another already made nucleotide Template Strand: the strand of DNA being copied Daughter Strand: the copied DNA strand DNA polymerase 1 can add a brand new nucleotide to the 31 end of theone that is already present Comes in the form of a nucleotide triphosphatea. dTTP, dATP, dCTP, dGTP (deoxythymine triphosphate etc.) Build up polynucleotide by adding one nucleotide at a time using DNA polymerase 1 When nucleotide is added to the 3 prime end of the nucleotide that is already there, 2 phosphates break off and one bonds to the hydroxyl group, typical dehydration synthesisii. 5 1  3 1 Exonuclease Ability  Like “Paccman” Removes on nucleotide at a time from the 5 prime phosphate end Removes the nucleotide with a hydrolytic reaction (where you add H2Oand break two monomers apart) Used to remove primersiii. 3 1  5 1 Exonuclease Ability  Can begin at the 3 prime hydroxyl and bite off one nucleotide at a time Functions as a “proof reader” Ex: DNA polymerase 1 tried to bond a C with an A instead of a T (violation of base pairing rules), if this happens, the enzyme uses 3151 exonuclease ability to remove the last nucleotide added to the 3prime end, it clips it off and the right one is put in its place4. DNA Polymerase III : FASTa. Two Activitiesi. 5 1  3 1 Polymerase Ability  Allows the addition of new nucleotides to the 31 hydroxyl end of another already made nucleotide Same as DNA Polymerase Iii. 3 1  5 1 Exonuclease Ability  “Proof reader” Same as DNA Polymerase Ib. DOES NOT HAVE 51  31 Exonuclease Abilityc. DNA Polymerase adds a nucleotide to the 3 Prime Hydroxyl end of another nucleotide that is already there5. Primase : “getting things started”a. Puts in a short RNA Primerb. If the DNA is AT the RNA is UA and the RNA primer is 5-10 nucleotides long and it “primes the pump”c. When the primer is put in, DNA Polymerase I or III can start adding nucleotides one at a time using the 53 polymerase ability6. DNA Ligase: the enzyme that can bond one nucleotide to the nucleotide next to it during DNA replication through dehydration synthesisa. Two nucleotides next to each other, release water, form covalent bond between a phosphate and hydroxyl group Replication Process:o Two nucleotides are anti-parallel 5131 and 3151o Helicase enters the DNA at the replication fork breaking the bonds between the A’s, T’s, C’s, and G’s.o An RNA Primer has to be antiparallel to template strand (has U’s on it bc RNA)o DNA Polymerase one adds DNA to 31 end of the primero New polynucleotide being elongated into the replication fork as the helicase unzips the moleculeo Leading Strand - is elongated continuously, as long as the helicase is unzipping the DNA molecule, the DNA polymerase III can continue to elongate the daughter of the leading strandas long as it adds to the 31 endo Lagging Strands - are elongated discontinuously Slower, because of the primers have to be antiparallel to the template strand, if one end of the nucleotide is the 51 end then the primer has to be 31.  The primer will be elongated one at a time at the 31.  Each segment is called and Oaksaki Fragment (100-300 nucleotides in length) DNA Polymerase I is going to come in and use its 5131 exonuclease ability to remove the primers It goes in and attacks the 51 end of the primer, it will digest primer, then use its polymerase ability to elongate the 31 end to connect to the 51 of the following fragments One nucleotide is bonded to the next by DNA ligaseo Primers are added until the helicase is finished unzipping and runs into the other helicaseo MAKE FLASHCARDS AND BE ABLE TO TELL WHAT ALL THE ENZYMES DO. ALSO TAKEA DNA MOLECULE LIKE HE DID AND DO IT BY PUTTING IN THE ORIENTATION (ANTIPARALLEL) AND SHOW HOW YOU’D ELONGATE THE STRANDS (LAGGING AND LEADING).o At the end of the DNA molecule, DNA polymerase I using its 5131 exonuclease ability will come in and digest out a nucleotide. There is no way fill in the gap once the primer at the end of the strand has been removed, we would need another primer on the outside but we cant build without a template molecule to attach it to. So what happens is, every time a chromosome is copied on the lagging strand there is gap left which cant be filled in because there is no 31 end to attach the nucleotides to. o Eukaryotes had linear chromosomes and at each end of the chromosome, every time a chromosome is replicated, it will get shorter by the length of a primer because we cant fill in the gap. They get shorter and shorter until eventually they start to eat away at important genes in your chromosomes. This problem is solved by the addition of telomeres.o Telomeres: is a sequence of DNA (TTAAGGG) beginning at the 5 prime end. It is found at the end of a chromosome and is this sequence in length, which is repeated between 100-300 times. As the chromosome gets shorter with each replication, they get shorter and so do the telomere, they form a buffer so the important genes are not removed(help?: http://www.youtube.com/watch?v=OKBVDCpAipU