BIOL 1441 1st Edition Lecture 29 Outline of Last Lecture I. DNAII. TransformationIII. DNA replication modelOutline of Current Lecture I. DNA replication modelII. DNA replicationIII. Repairing DNACurrent LectureI. DNA replication modela. Semi-conservative modeli. 2 strands of the parental molecule separate and each functions as a template for synthesis of a new, complementary strandii. CORRECT MODELb. Competing models:i. Conservative model: 2 parental strands reassociate after acting as templates for new strands, restoring the parental double helixii. Dispersive model: each strand of both daughter molecules contains a mix of old and newly synthesized DNAII. DNA Replicationa. 6 billion base pairs (in each cell), takes a few hrs to copyb. Very few errors- 1 in 10 billion nucleotidesc. Copying of DNA is remarkable in its speed and accuracyd. More than a dozen enzymes and other proteins participate in DNA replicatione. Different in prokaryotes than in eukaryotes f. Begins at origins of replication- 2 DNA strands are separated, opening up a replication “bubble”i. Prokaryotes- single origin of replicationThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.ii. Eukaryotes- may have hundreds of origins of replicationg. Proceeds in both directions from each origin, until the entire molecule is copiedh. At the end of each replication bubble is a replication fork, a Y-shaped region where new DNA strands are elongatingi. Elongating a New DNA Strandi. DNA polymerase- enzyme that catalyzes elongation of new DNA at a replication forkii. Rate of elongation ~500 nucleotides per second in bacteria & 50 per second in human cellsiii. Bacteria- DNA polymerase I & IIIiv. Humans- at least 11 different DNA polymerases!v. Actually adding nucleotide triphosphatevi. ATP (adnosine triphosphate)- except sugar in ATP is ribose, not deoxyribosej. Triphosphate Nucleotidesi. Highly reactive- triphosphate tails have unstable cluster of negative chargeii. As each nucleotide is added to growing DNA chain, it loses 2 phosphate groups forming pyrophosphateiii. Subsequent hydrolysis of pyrophosphate into 2 molecules of inorganic phosphate- exergonic rxn, drives polymerization rxn of growing DNA chaink. Antiparallel Elongationi. 2 strands oriented in opposite directions- affects replicationii. DNA polymerase can only add nucleotides to the free 3’ end of a growing strand1. Can only move 1 way down strandl. A new DNA strand can only elongate in the 5’ to 3’ directionm. Only orientation DNA polymerase can add basesn. How does the 2nd strand (3’–5’) replicate?i. Replication is discontinuousii. DNA polymerase jumps ahead, elongates in segmentso. Lagging strand replication:i. Leading strand-DNA polymerase adds DNA continuously, moving toward the replication forkii. Lagging strand-DNA polymerase must work in direction away from replication fork1. Lagging strand- synthesized as a series of segments -Okazaki fragments2. Joined together by DNA ligase (glues them)p. Priming DNA Synthesisi. DNA polymerase cannot initiate synthesis of a polynucleotide1. Can only add nucleotides to 3 ¢ endii. Need an RNA primer- short segment of RNAiii. Primase- start an RNA chain from scratch1. Binds primer and begins replicationiv. Leading strand- one primer v. Lagging strand- each Okazaki fragment must be primed separatelyq. Other Proteins That Assist DNA Replicationi. Helicase - untwists double helix & separates the template DNA strands at the replication forkii. Single-strand binding protein- binds & stabilizes single-stranded DNA until used as templateiii. Topoisomerase- corrects “overwinding” ahead of replication forks by breaking, swiveling, & rejoining DNA strandsiv. Primase- synthesizes an RNA primer at the 5¢ ends of the leading strand and the Okazaki fragmentsv. DNA pol III- continuously synthesizes the leading strand and elongates Okazaki fragmentsvi. DNA pol I- removes primer from the 5¢ ends of the leading strand and Okazaki fragments, replacing primer with DNA and adding to adjacent 3¢ ends vii. DNA ligase- joins the 3¢ end of the DNA that replaces the primer to the rest of the leading strand & joins lagging strand fragmentsIII. Repairing DNAa. DNA polymerase- proofread newly made DNA, replacing any incorrect nucleotidesb. DNA damage- oxygen radicals, radioactivity, X-rays, UV light, spontaneous changes under normal conditionsc. Cells monitor & repair DNA continuouslyi. 130 repair enzymes in humansd. Mismatch Repairi. Enzymes correct errors in base pairinge. Thymine dimeri. Covalent bonding of two adjacent thymine residues within a DNA molecule, often catalyzed by UV radiation or chemical mutagenic agentsf. Nucleotide excision repairi. Enzymes but out and replace damages stretches of DNAg. Replicating the Ends of DNA Moleculesi. Eukaryotic DNA- telomeres at ends of nucleotide sequences, repetitive short nucleotide sequenceii. Telomeres- do not prevent the shortening of DNA molecules, they postpone erosion of genes near the ends of DNA moleculesh. Errors in replication:i. Polymerase adds wrong nucleotide- if mistake gets through can cause mutationii. Cancer1. Normal shortening of telomeres protects body from cancer- limits number of cell divisions somatic cells can undergo2. More cell divisions- more DNA replication- more errors3. Cancer cells have telomerase activity- lengthen telomeres, keep dividing- immortala. Also keep acquiring more genetic