Chapter 9: DNA structure and analysis

1. Chapter 9: DNA structure and analysis

2. Where we’re going • The Central Dogma- DNA makes RNA makes Protein • Evidence for DNA= genetic material • Structure of DNA- key words of polarity, complementary, antiparallel, knowing the bases.

3. CENTRAL DOGMA OF MOLECULAR GENETICS: • DNA makes DNA (replication) • DNA makes (carries the information to make) • RNA (transcription); • RNA makes (carries the information to make) • PROTEIN (translation) which contributes to a particular • TRAIT • Fig. 9-1

4. Convincing you that DNA is the genetic material • Early- DNA=boring, wrong, mislead a generation of scientists- just a scaffold for the more interesting proteins- a repeating A-T-C-G structure. • Then, Chargaff- A=T, G=C, but the amounts of each type could vary quite a bit. • I. DNA as the genetic material: Avery, and Hershey/Chase,: • Avery showed that what Griffith found, transformation of rough Streptococcus ‑‑‑‑>smooth, was caused by DNA. Fig 9-2,3 • The smooth phenotype was due to the presence of a capsule- made Strep pneumoniae resistant to phagocytosis.

5. DNA as the genetic material: Avery, and Hershey/Chase: • I. Avery showed that what Griffith found, transformation of rough Streptococcus ‑‑‑‑>smooth, was caused by DNA. Fig 9-2,3 • The smooth phenotype was due to the presence of a capsule- made Strep pneumoniae resistant to phagocytosis.

8. Hershey-Chase • Bacteriophage infect & kill cells (9-5) • Known to inject material into cells- not the whole virus- so the injected material had the information to make a virus. • Protein or DNA??? • It was DNA, and the work done after Watson-Crick, so they got more press

10. II. Structure of DNA: Linear, double‑strandedantiparallelcomplementary chains of deoxynucleotides: • deoxynucleotides: • nitrogenous bases: Adenine, thymine, cytosine, guanine; A=T, G=C; • The chains have polarity because of the linkage‑ a 5' and 3' end to each molecule. • Learning Objectives(LO): Be able to recognize the bases found in DNA and RNA, and the partners to which they pair. • Distinguish between a nucleoside & nucleotide

11. 1 ring, big name 2 rings, small name

12. Base pairing- 2 & 3 H-bonds Great figure! From Ch. 14, sort of Q: What difference does it make if you have 2 or 3 H-bonds?

13. The nucleotides are more commonly called nucleoside monophosphates.

14. Learning Objective: What is the structure of DNA? Specifically: • Understand the polarity of DNA and its antiparallel nature: • given a sequence, be able to give its complementary sequence. • Be able to identify the major and minor grooves of DNA. • Know what is meant by DNA being a right-handed helix, and by • supercoiling. • Be able to identify the coding and anticoding strands of DNA • when it is used as a template for transcription.

15. Nucleotides are linked together- POLARITY!

16. Six mistakes! Find 3!

17. Six mistakes! Find 3!

18. 2 nm 3.4 nm, 0.34 nm/base 10 bp/turn Rt handed dbl helix, complementary, antiparallel

19. Supercoiled!

20. How I test this: • 5' ATGACCTTAGG3‘ • - give the complementary strand, RNA or DNA

21. Features that make DNA suitable as genetic material: • a. Strands are complementary: thus, each strand is the template, holds the information, for the other strand; the pattern for replication is built‑in. • b. The bases allow for a three‑base code: there are 64 possible combinations of three bases, more than enough to code for all the amino acids. • c. The structure allows for both faithful duplication, and for mutations that will then be perpetuated.

22. Structure of RNA: • Ribose and uracil, not deoxyribose and thymine. • usually single stranded • can fold, to produce secondary structure • Three main types, but there are others: rRNA, mRNA, tRNA

23. III. Neat things you can do with DNA, and what it means • A. Denaturation and renaturation: DNA can be denatured- rendered SS- by heat or NaOH treatment. HOWEVER: upon heating to 68C, it will renature- find its complementary bases and reform DS DNA. • B. hybridization: Because DNA can renature, you can prepare probes: labeled DNA that will hybridize to unlabeled target DNA, either in solution, or when the target is immobilized to paper. A probe will find its complementary DNA rapidly, even in the midst of a vast excess of non-target DNA. One application: FISH (9-15).

25. Quiz (ignore for now) • Something like last week’s meiosis question • Central Dogma- major terms • Sequence-complement • Recognize a purine nucleotide • How does meiosis explain segregation and independent assortment????

26. C. Determining its size • Gel electrophoresis: small DNA fragments migrate faster than large; rate is an inverse log function. Fig 10-19 • Pulsed field gel electrophoresis: variation of electrophoresis that can separate large fragments of DNA. (not a test Q!) • Electron microscopy: Opening figure of Ch. 10

28. What to know: • See the learning objectives! But specifically: • Evidence for DNA being genetic material: Avery & Hershey/Chase • Structure (including components), w/ key words (rt handed helix, complementary, polarity, antiparallel, etc.) • Denaturation, hybridization, size determination.

29. Chapter 10- DNA replication & recombination • Check out the “how do we know” section • I. Replication is: • SEMICONSERVATIVE • BIDIRECTIONAL • SEMIDISCONTINUOUS

30. A. Semiconservative: Fig. 10.3, 10.4 • Proven by Messelsohn‑Stahl experiment: Heavy (15N) DNA‑‑‑‑‑> HL, then LL, • with a fixed amount of HL remaining. Separation is by CsCl density gradient centrifugaton. (Also Taylor, Woods, Hughes)

33. B. BIDIRECTIONAL: Figs 10-6; Low 3H thymidine pulse is followed by high 3H thymidine pulse; results after autoradiography is a set of symmetrical dark bands. • Bacterial spores- germinate, resulting in synchronous initiation of DNA replication pulse with low 3H thymidine pulse high 3H thymidine pulse autoradiography. The results show symmetrical lines of thymidine incorporation into the DNA: (See Q. 32 in your text)

36. C. Semidiscontinuous: Fig. 11.11 One strand is made continuously, one strand discontinuously • We’ll cover evidence a bit later.

38. II. Process of replication: AS WITH ALL MACROMOLECULAR SYNTHESIS: INITIATION, ELONGATION, TERMINATION. • Cool video of the process- an animation: • http://www.wehi.edu.au/education/wehi-tv/dna/replication.html • http://www.youtube.com/watch?v=4jtmOZaIvS0&feature=related

39. http://www.youtube.com/watch?v=gL3aigv7 • http://www.youtube.com/watch?NR=1&v=teV62zrm2P0w4A&feature=related

40. The process of DNA replication is driven by 1) antiparallel nature of DNA 2) the nature of DNA polymerases: a) ONLY elongate from a 3'-OH, i.e., only replicate in a 5'-3'direction; DO NOT initiate, ONLY elongate. • Bacterial DNA polymerases are VERY fast: 1000 bp/sec! • 5'---------------T3'OH + pppdG3'OH • 3'---------------ACGGATCGAGAG-----------------5' • 5'---------------TG3'OH +pppdC3'OH • 3'---------------ACGGATCGAGAG-----------------5' • 5'---------------TGC3'OH + pppdC3'OH • 3'---------------ACGGATCGAGAG-----------------5' • etc.

42. A. INITIATION: BEGINS at a particular location, the origin‑ signaled by the cell. Prokaryotes have a single origin, eukaryotes have many. Replication is initiated by an increase in cell mass, triggered by signals received from the cell. Initiation proteins open up the helix at the origin. Key protein: Dna A. Fig. 10-9. A region replicated by a single origin is a replicon. Bacteria usually have one, euk. have many hundreds of replicons.

43. Origin region A helicase and loader of primase

45. B. ELONGATION: once synthesis has begun, it usually proceeds bidirectionally. Because synthesis is always 5'‑3', synthesis tends to be CONTINUOUS on one strand of a replication fork, and DISCONTINUOUS on the other strand of the fork. Primase, with the help of the mobile promoter & helicase, theDNA BC complex, moves down the lagging strand, laying down primer for DNA pol to use. Lagging strand synthesis produces short fragments of 1-2K bases, called Okazaki fragments. (Fig 10-11)

47. C. HOW WE KNOW THIS: Labeling experiments with replicating viruses; if you label replicating DNA with a short pulse of radioactivity, you will label short (Okazaki) fragments, and longer continuous strand fragments, that can be separated by an alkaline sucrose density gradient (crude blackboard drawing here)

48. D. Other players: Helicase: again, the DnaBC complex helps primase get started, and also separates the strands to allow replication; DNA gyrase: Introduces negative supercoils, acting to allow the DNA to “swivel”, preventing overwinding of the helix. There’s also a single-stranded binding protein (SSBP) that, well, binds single stranded DNA- keeping it SS as needed.

49. E. Completing the job: the problem of ends. • Fig. 11-16, 17: circles do not present a problem for termination; two circles are made. Linear DNA does present a problem, b/c of the gap left by the lack of primer at the 5' end of the new DNA. In Eukaryotes, the problem is solved by TELOMERASE.