Unit 6 1 UNIT 6 PART A: DNA STRUCTURE PART B: DNA REPLICATION PART A: DNA STRUCTURE The most remarkable of al the properties of living cels and organisms is their ability to reproduce themselves with nearly perfect fidelity for countles generations. This continuity of inherited traits implies constancy, over thousands or milions, of years in the structure of the molecules that contain the genetic information. This hereditary information is preserved in DNA. DNA is a long thin organic polymer in which the linear sequence of covalently linked nucleotide subunits encodes the genetic mesage. Two of these polymeric strands are twisted about each other to form the DNA double helix, in which each monomeric subunit in one strand pairs specificaly with the complementary subunit in the opposite strand. This unit provides an overview of the structure of nucleotides and nucleic acids found in most cels. Asignment: Nelson & Cox, pp. 271 - 281, 287 - 288, 945 - 971, 976. 1. Given the structures of the four common bases (Fig. 8-2, p. 272), identify each as a purine or pyrimidine and give its common name. (Note: You wil not be asked to draw the structure of the bases, given the name.) Unit 6 2 2. Nucleosides and Nucleotides a. Given the structure of the bases in Fig. 8-2 (p. 272), draw structures representing (Fig. 8-1, p. 271): 1) a 2'-deoxynucleoside 2) a 2'-deoxynucleotide 3) a 2'-deoxynucleoside 5'-di- or triphosphate b. Use Fig. 8-1 (p. 271) to discuss the numbering convention for the pentose ring. Which atom in the sugar is the base atached to? What kind of bond joins the sugar to the base (p. 272)? Using Fig. 8-4 (p. 273), identify the atom that is found at the 2' position in deoxyribonucleotides? - in ribonucleotides? What functional group is at the 3' position in ribo- and deoxy-ribonucleotides? To which atom in the sugar is the phosphate bound? c. Use Fig. 8-4 (p. 273) to discuss the nucleotides found in DNA. Compare and contrast these nucleotides to those found in RNA. 3. Draw a section of DNA (use the leters A, C, G, and T to represent the structure of the base). Point out the following (Fig. 8-7, p. 275): a. phosphodiester bond (or linkage; note the diference betwen a phosphodiester bond and a phosphanhydride bond by comparing to Fig. 1-15, p. 12) b. sugar-phosphate backbone c. 5' and 3' ends d. overal charge 4. Distinguish betwen an oligonucleotide and a polynucleotide (p. 276). 5. Use Fig. 8-11 (p. 277) to discuss the hydrogen bonds that stabilize the DNA double helix. Unit 6 3 6. How was DNA shown to be the genetic material (p. 278)? Why was the base composition (Chargaf's rules) important in suggesting a model for the structure of DNA (p. 278)? What other information was crucial in the structure determination (p. 278)? 7. The structure of B-form DNA (you are not responsible for the other forms of DNA described on p. 281) a. Use Fig. 8-13 (p. 279) to discuss the structure of double-stranded DNA. Point out the major and minor grooves, the sugar-phosphate backbones, and the stacked base pairs. b. Use Fig. 8-14 (p. 279) to discuss the strand polarity in double- stranded DNA. What word is used to describe the polarity of the two strands? What base pairs are found in DNA? Discuss the key diference betwen these two base pairs. What is the position of the sugar-phosphate backbone relative to the bases? c. What two forces stabilize the DNA double helix (p. 279)? 8. Do problem 8-1 on base pairing in DNA (p. 280). 9. Double-stranded DNA can be reversibly denatured and renatured (pp. 287 - 288). a. Discuss the types of bonds broken during denaturation (p. 287). b. Use Fig. 8-27 (p. 288) to discuss the following: 1) What is the melting point (t m )? 2) What is the relationship betwen t m and the base composition? c. Define what is meant by "annealing" (p. 287). Unit 6 4 10. Define and distinguish betwen DNA replication, transcription, and translation (p. 945). 11. What is a gene (p. 948)? How are genes named (p. 976)? Later you wil learn about sequences that control expresion of genes. These and introns are part of the modern definition of a gene. 12. What distinguishes a plasmid from a chromosome (p. 949)? 13. Is al eukaryotic DNA stored in the nucleus (p. 951)? Explain. 14. Eukaryotic chromosomes are complex. Define the following (pp. 952 - 953): a. intron b. exon c. satelite DNA d. centromere e. telomere 15. DNA Supercoiling a. What is meant by "linking number" (p. 956; do not worry about the equations)? What is a "topoisomer" (p. 957)? b. Discuss how over-winding or underwinding the DNA double helix leads to the formation of positive or negative supercoils respectively (Figs. 24-14 and 24-17; pp. 955, 957). c. List two reasons why it is advantageous for cels to maintain their DNA in the underwound state (p. 956). Unit 6 5 d. What is a topoisomerase (p. 958)? 1) Diferentiate betwen type I and type I topoisomerases in E. coli (p. 958). 2) Describe how E. coli topoisomerase I removes negative supercoils, thereby increasing the linking number (Fig. 24-21, p. 959). 16. Use Figs. 24-26 (p. 963) and 24-27 (p. 964) to discuss the packaging of DNA in eukaryotes into nucleosomes. What is unique about the amino acid composition of histone proteins that is crucial to their DNA-binding function (p. 963)? 17. Use Figs. 24-29 (p. 965), 24-30 (p. 967), 24-32 (p. 968), and 24-33 (p. 968) to discuss higher order organization of eukaryotic DNA. Unit 6 6 PART B: DNA REPLICATION Long before the structure of DNA became known, scientists wondered how organisms create copies of themselves and how cels produce many identical copies of large and complex macromolecules. The proces of DNA replication provided the first biological example of the use of a molecular template to guide the synthesis of macromolecules. The properties of DNA replication and the mechanisms used by the enzymes that catalyze it are esentialy identical in al organisms. In the following section, we wil study the molecular details of DNA replication. Asignment: Nelson & Cox, pp. 289 - 292, 975 - 991, 996 - 997, 1053 - 1056 (stop at "Some viral RNAs.."). 1. Explain what is meant by semi-conservative replication (p. 977). 2. Define what is meant by the term origin of replication (p. 978). 3. What is a replication fork (p. 978)? Use Fig. 25-3 (p. 978) to discuss bi-directional replication. 4. Using Fig. 25-4 (p. 979), discuss leading and lagging strand synthesis. What is the direction of DNA synthesis? In what direction is the template strand read? Which strand is replicated continuously, and which discontinuously? What are Okazaki fragments? 5. Distinguish betwen an exonuclease and an endonuclease (p. 979). Unit 6 7 6. DNA Polymerases a. What are the two "central requirements" of DNA polymerase for DNA polymerization (pp. 979 - 980)? b. Use Fig. 25-5 (p. 980) to discuss the reaction catalyzed by DNA polymerases. 1) Which type of nucleotide (dNMP, dNDP, or dNTP) are used as substrates? 2) What proces drives the reaction toward the formation of products (p. 979)? Discuss in terms of Le Chatelier's principle. c. Define procesivity (p. 980). d. Use Fig. 25-7 (p. 981) to discuss proofreading by DNA polymerase. Which exonuclease activity is responsible for DNA proofreading (pp. 980 - 981)? e. Use Table 25-1 (p. 982) to compare DNA pol I and DNA pol II. Pay particular atention to the following: 1) 3' to 5' exonuclease activity (proofreading) 2) 5' to 3' exonuclease activity 3) Rates of polymerization 4) Procesivity f. Use Fig. 25-10 (p. 984) to explain why DNA pol II is more procesive than DNA pol I. Unit 6 8 7. Discuss the function of each of the following enzyme types in DNA replication (p. 984): a. helicase b. topoisomerase c. DNA-binding proteins d. primase e. DNA ligase 8. Initiation of Replication a. Use Fig. 25-11 (p. 985) to discuss the sequence features at the origin of replication. (Note: Do not concern yourself with actual sequences, just the number and diferent types of repeated sequences present!) b. Use Fig. 25-12 (p. 985) to discuss the events that initiate DNA replication 1) What is the function of the R and I sites? What happens at these sites that leads to unwinding of the double helix at the DUE (p. 985)? 2) What is the role of the helicase that enters next (p. 986)? 9. Elongation (pp. 987 - 989) a. Using Fig. 25-13 (p. 987) as a guide, discuss the following: 1) As the fork enlarges, what enzyme continues to be involved in unwinding the double helix? 2) What enzyme counteracts the positive supercoiling caused by the helicase, by introducing negative supercoils? Unit 6 9 3) What protein binds to the resulting single-stranded DNA? Why is this important? 4) What proces is required before DNA synthesis can occur on the single-stranded template? Explain. What enzyme caries this out? hy is RNA used as a primer instead of DNA? 5) What enzyme now caries out the bulk of DNA replication? b. Using Fig. 25-14 (p. 988) as a guide, discuss how a dimer of DNA polymerase at the replication fork can copy both parental strands simultaneously. Discuss the role of the beta subunit. c. Name the enzyme that removes the RNA primer (p. 989). Which activity from table 25-1 (p. 982) is used for RNA primer removal? d. Name the enzyme that then catalyzes the formation of phosphodiester bonds betwen the newly synthesized DNA fragments (p. 989). 10. DNA replication is bi-directional. Use the diagram below to explain bi-directional DNA replication from a single origin of replication (represented by the dots in the middle of the single-stranded region). Use squiggled lines to represent RNA primers and straight lines for DNA. Use arow heads pointing from 5' to 3' to denote the direction of polynucleotide synthesis. 5' 3' 5' 3' Unit 6 10 11. Telomeres (pp. 1053 - 1056) a. What are telomeres (pp. 1053 - 1054)? b. Discuss why linear chromosomes can not be entirely copied by lagging strand synthesis (p. 1054). b. Name the enzyme that adds telomeric ends (p. 1054). What type of enzyme is it? c. Use Fig. 26-39 (p. 1055) to discuss telomere addition. What serves as the primer for telomere addition? What serves as the template? d. In humans, what types of cels (germline or somatic) have telomerase (p. 1055)? e. Discuss the role of telomeres in cel senescence (p. 1055). 12. Base-excision Repair (pp. 289 - 292, 996 - 997) a. Use Fig. 8-30a (p. 290) to discuss the spontaneous cytosine deamination reaction. b. Use Fig. 25-25 (p. 997) to discuss base-excision repair, naming each enzyme and discussing the function of each. 13. List thre key proceses that contribute to the high fidelity of DNA replication (p. 992, summary). Jim Blankenship Microsoft Word - U06_F08.doc
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