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Ch. 16 Warm-Up 1. Draw and label a nucleotide. 2. Why is DNA a double helix? 3. What is the complementary DNA strand to: DNA: A T C C G T A T G A A C Ch. 16 Warm-Up 1. What was the contribution made to science by these people: A.Hershey and Chase B.Franklin C.Watson and Crick 2. Chargaff’s Rules: If cytosine makes up 22% of the nucleotides, then adenine would make up ___ % ? 3. Explain the semiconservative model of DNA replication. Ch. 16 Warm-Up 1. What is the function of the following: A. Helicase B. DNA Ligase C. DNA Polymerase (I and III) D. Primase E. Nuclease 2. How does DNA solve the problem of slow replication on the lagging strand? 3. Code the complementary DNA strand: 3’ T A G C T A A G C T A C 5’ 4. What is the function of telomeres? THE MOLECULAR BASIS OF INHERITANCE Chapter 16 What you must know The structure of DNA. The major steps to replication. The difference between replication, transcription, and translation. The general differences between the bacterial chromosome and eukaryotic chromosomes. How DNA is packaged into a chromosome. Problem: Is the genetic material of organisms made of DNA or proteins? Frederick Griffith (1928) Frederick Griffith (1928) Conclusion: living R bacteria transformed into deadly S bacteria by unknown, heritable substance Oswald Avery, et al. (1944) Discovered that the transforming agent was DNA Their conclusion was based on experimental evidence that only DNA worked in transforming harmless bacteria into pathogenic bacteria Many biologists remained skeptical, mainly because little was known about DNA Hershey and Chase (1952) Bacteriophages: virus that infects bacteria; composed of DNA and protein Protein = radiolabel S DNA = radiolabel P Hershey and Chase (1952) Conclusion: DNA entered infected bacteria DNA must be the genetic material! Edwin Chargaff (1947) Chargaff’s Rules: DNA composition varies between species Ratios: %A = %T and %G = %C Rosalind Franklin (1950’s) Worked with Maurice Wilkins X-ray crystallography = images of DNA Provided measurements on chemistry of DNA The width suggested that the DNA molecule was made up of two strands, forming a double helix. James Watson & Francis Crick (1953) Discovered the double helix by building models to conform to Franklin’s X-ray data and Chargaff’s Rules. James Watson & Francis Crick (1953) Watson and Crick built models of a double helix to conform to the X-rays and chemistry of DNA Franklin had concluded that there were two antiparallel sugar-phosphate backbones, with the nitrogenous bases paired in the molecule’s interior At first, Watson and Crick thought the bases paired like with like (A with A, and so on), but such pairings did not result in a uniform width Instead, pairing a purine with a pyrimidine resulted in a uniform width consistent with the X-ray LE 16-UN298 Purine Purine++purine: purine:too toowide wide Pyrimidine + pyrimidine: too narrow Pyrimidine + pyrimidine: too narrow Purine + pyrimidine: width consistent with X-ray data Structure of DNA DNA = double helix “Backbone” = sugar + phosphate “Rungs” = nitrogenous bases By the 1950s, it was already known that DNA is a polymer of nucleotides, each consisting of a nitrogenous base, a sugar, and a phosphate group Structure of DNA Nitrogenous Bases Adenine (A) Guanine (G) Thymine (T) Cytosine (C) purine pyrimidine Pairing: purine + pyrimidine A=T GΞC Structure of DNA Hydrogen bonds between base pairs of the two strands hold the molecule together like a zipper. Covalent bonds connect the nucleotide components together Structure of DNA Antiparallel: one strand (5’ 3’), other strand runs in opposite, upside-down direction (3’ 5’) DNA Comparison Prokaryotic DNA Double-stranded Circular One chromosome In cytoplasm No histones Supercoiled DNA Eukaryotic DNA Double-stranded Linear Usually 1+ chromosomes In nucleus DNA wrapped around histones (proteins) Forms chromatin Problem: How does DNA replicate? Replication: Making DNA from existing DNA 3 alternative models of DNA replication Meselson & Stahl Meselson & Stahl Replication is semiconservative – “daughter” DNA molecules consist of one original parental strand and one new strand DNA Replication Video http://www.youtube.com/watch?v=4jtmOZaIvS0 &feature=related Major Steps of Replication: 1. Helicase: unwinds DNA at origins of replication a. Initiation proteins separate 2 strands forms replication bubble 2. Primase: puts down RNA primer to start replication 3. DNA polymerase III: adds complimentary bases to leading strand (new DNA is made 5’ 3’) 4. Lagging strand grows in 3’5’ direction by the addition of Okazaki fragments 5. DNA polymerase I: replaces RNA primers with DNA 6. DNA ligase: seals fragments together 1. Helicase unwinds DNA at origins of replication and creates replication forks Getting Started: Origins of Replication Replication begins at special sites called origins of replication, where the two DNA strands are separated by helicase, opening up a replication “bubble” A eukaryotic chromosome may have hundreds or even thousands of origins of replication Replication proceeds in both directions from each origin, until the entire molecule is copied At the end of each replication bubble is a replication fork, a Y-shaped region where new DNA strands are elongating 2. Primase adds RNA primer Priming DNA Synthesis DNA polymerases cannot initiate synthesis of a polynucleotide; they can only add nucleotides to the 3 end The initial nucleotide strand is a short one called an RNA or DNA primer An enzyme called primase can start an RNA chain from scratch 3. DNA polymerase III adds nucleotides in 5’3’ direction on leading strand Elongating a New DNA Strand Enzymes called DNA polymerase III catalyze the elongation of new DNA at a replication fork Each nucleotide that is added to a growing DNA strand is a nucleoside triphosphate The rate of elongation is about 500 nucleotides per second in bacteria and 50 per second in human cells Replication on leading strand Leading strand vs. Lagging strand 4. DNA polymerase III adds nucleotides in 3’5’ direction on lagging strand Only one primer is needed to synthesize the leading strand, but for the lagging strand each Okazaki fragment must be primed separately LE 16-15_1 Primase joins RNA nucleotides into a primer. 3 5 5 3 Step 1: Primase joins RNA nucleotides into a primer Template strand Overall direction of replication LE 16-15_2 3 Primase joins RNA nucleotides into a primer. 5 5 Template strand 3 3 DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. RNA primer 5 Overall direction of replication 3 5 Step 2: DNA pol IIl adds DNA nucleotides to the primer, forming an Okazaki fragment LE 16-15_3 Primase joins RNA nucleotides into a primer. 3 5 5 Template strand 3 3 DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. RNA primer 3 5 5 After reaching the next RNA primer (not shown), DNA pol III falls off. Okazaki fragment 3 3 5 5 Overall direction of replication Step 3: After reaching the next RNA primer (not shown), DNA pol III falls off. LE 16-15_4 Primase joins RNA nucleotides into a primer. 3 5 5 Template strand 3 3 DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. RNA primer 3 5 5 After reaching the next RNA primer (not shown), DNA pol III falls off. Okazaki fragment 3 3 5 5 After the second fragment is primed, DNA pol III adds DNA nucleotides until it reaches the first primer and falls off. 5 3 3 5 Overall direction of replication Step 4: After the second fragment is primed, DNA pol III adds DNA nucleotides until it reaches the first primer and falls off. LE 16-15_5 Primase joins RNA nucleotides into a primer. 3 5 5 3 Template strand 3 DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. RNA primer 3 5 5 After reaching the next RNA primer (not shown), DNA pol III falls off. Okazaki fragment 3 3 5 5 After the second fragment is primed, DNA pol III adds DNA nucleotides until it reaches the first primer and falls off. 5 3 3 5 5 3 DNA pol I replaces the RNA with DNA, adding to the 3 end of fragment 2. 3 5 Overall direction of replication Step 5: DNA pol I replaces the RNA with DNA, adding to the 3 end of fragment 2. LE 16-15_6 Primase joins RNA nucleotides into a primer. 3 5 5 3 Template strand 3 DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. RNA primer 3 5 5 After reaching the next RNA primer (not shown), DNA pol III falls off. Okazaki fragment 3 3 5 5 After the second fragment is primed, DNA pol III adds DNA nucleotides until it reaches the first primer and falls off. 5 Step 6: DNA ligase forms a bond between the newest DNA and the adjacent DNA of fragment 1. 3 3 5 5 3 Step 7: DNA pol I replaces the RNA with DNA, adding to the 3 end of fragment 2. 3 5 DNA ligase forms a bond between the newest DNA and the adjacent DNA of fragment 1. The lagging strand in the region is now complete. 5 3 3 5 Overall direction of replication The lagging strand in the region is now complete. Okazaki Fragments: Short segments of DNA that grow 5’3’ that are added onto the Lagging Strand DNA Ligase: seals together fragments Proteins That Assist DNA Replication Helicase untwists the double helix and separates the template DNA strands at the replication fork Single-strand binding protein binds to and stabilizes single-stranded DNA until it can be used as a template Topoisomerase corrects “overwinding” ahead of replication forks by breaking, swiveling, and rejoining DNA strands Proteins That Assist DNA Replication Primase synthesizes an RNA primer at the 5 ends of the leading strand and the Okazaki fragments DNA pol III continuously synthesizes the leading strand and elongates Okazaki fragments 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 DNA ligase joins the 3 end of the DNA that replaces the primer to the rest of the leading strand and also joins the lagging strand fragments Concept Check 1. 2. 3. 4. 5. Which of the following best describes the arrangement of the sides of the DNA molecule? A. twisted B. antiparallel C. bonded D. alternating If a DNA molecule is found to be composed of 40% thymine, what percentage of guanine would be expected. A. 10% B. 20% C. 40% D. 80% The enzymes that break hydrogen bonds and unwind DNA are: A. primers B. forks C. helicases D. polymerases During replication, what enzyme adds complimentary bases? A. helicase B.synthesase C. replicase D. polymerase 6. The base pair rules states that: A. Replication is semiconservative B. A pairs with T, G pairs with C C. DNA is a double helix held together by hydrogen bonds D. A pairs with G, T pairs with C Proofreading and Repair DNA polymerases proofread as bases added Mismatch repair: special enzymes fix incorrect pairings Nucleotide excision repair: Nucleases cut damaged DNA DNA poly and ligase fill in gaps Nucleotide Excision Repair Errors: Pairing errors: 1 in 100,000 nucleotides Complete DNA: 1 in 10 billion nucleotides Problem at the 5’ End DNA poly only adds nucleotides to 3’ end No way to complete 5’ ends of daughter strands Over many replications, DNA strands will grow shorter and shorter Telomeres: repeated units of short nucleotide sequences (TTAGGG) at ends of DNA Telomeres “cap” ends of DNA to postpone erosion of genes at ends (TTAGGG) Telomerase: enzyme that adds to telomeres catalyzes the lengthening of telomeres in eukaryotic germ cells Telomeres stained orange at the ends of mouse chromosomes Telomeres & Telomerase BioFlix: DNA Replication http://media.pearsoncmg.com/bc/bc_0media_b io/bioflix/bioflix.htm?8apdnarep