Announcements 1. Pick up lab overview for transformation lab this week. 2. Homework-problem set 4- due in lab this week. 3. Look over Ch. 11, problems 4, 5, and 8 for exam Group B presentations are coming up 10/29, 30; start thinking of topics and deciding on sources. Group A did very well - pressure is on! 5. Review session in class Wednesday. Bring your questions! 6. Exam 2 next week: 10/17, 18, and multiple choice and 7 written (exam 1 had 18 multiple choice and 9 written). Exam is at CLAS testing center, available 3 days this time. Hours are 9-9 Thursdays, 9-5 Fridays, and 3-7 Sundays. Bring a pencil, bluebook, calculator. 7. Summer scholar program - research opportunity in summer, $2400 stipend. Need to find a faculty member to sponsor you. Often need to volunteer in the lab spring semester. Application includes a formal written proposal; deadline mid-Feb.; need 56 credits completed by start of summer and be returning next fall to CMU.

Review of Last Lecture 1. Evidence that DNA is genetic material 2. Structure of DNA/RNA: 5 different bases, 2 different sugars, phosphates 3. History: the race to determine the structure of DNA was VERY competitive; 2 key pieces of data = Chargaff’s base compostion analysis and X-ray diffraction studies

Outline of Lecture 20 I. Structure of DNA II. Analytical analysis of nucleic acids III. Replication of DNA How is DNA organized? 1 single chain, 2 chains, 3 chains? How does the structure allow for replication, expression, storage and mutation?

I. The DNA Double Helix DNA structure Double helical –major, minor grooves –right-handed – bases are 3.4 Å apart (10 Å = 1 nm) –10 bases/turn Complementary Base Pairing –through H bonds: A=T, G  C Antiparallel Strands –5’ to 3’ –3’ to 5’ Discussion of original paper in class Friday

Right- and Left-handed DNA

Base-Pairing in DNA A=T GCGC

Structure of RNA Sugar: ribose, not 2-deoxyribose Bases: uracil, not thymine Organization: single-stranded, not double-stranded How is genetic information in DNA expressed? First step is transcribing RNA from DNA - single-stranded RNA is generated using DNA as a template

Reading DNA Strands Single strand of DNA: 5’-AGCATTCG-3’ 3’-TCGTAAGC-5’ Complementary strand of above, usually written 5’ to 3’: 5’-CGAATGCT-3’ Double-stranded fragment is written: 5’-AGCATTCG-3’ 3’-TCGTAAGC-5’

Learning Check The sequence of the dwarf gene in garden peas is as follows: 5’ - A G C T A C G T -3’ 3’ - T C G A T G C A -5’ Write the RNA sequence transcribed from the top strand of DNA, 5’- 3’.

Denaturation/Renaturation Which DNA has higher GC content and why? II. Analytical analyses of nucleic acids 1 2 Determining the Tm allows for an estimate of the base composition of a DNA sample

A C G C T T G C G A G G T T G G G C C A A C C C T T T G C G C A AA C G C G U U U G C G C Transcription of 1 strand of DNA 3 A C G C T T G C G A G G T T G G G C C A A C C C T T T G C G C A AA C G C G Heat - denature U U U G C G C A A A C G C G Add RNA to denatured DNA; allow to hybridize Hybrid A C G C T T G C G A G G T T G G G C C A A C C C Nucleic Acid Hybridization

Nucleic Acid Gel Electrophoresis

Base Pairing Rules What makes nucleic acids acidic?

Points to know about DNA structure Note how many hydrogen bonds are in the base pairing: –If 2, then the pair is AT –If 3, then the pair is GC –Recall that A and G are purines with 2 rings, while T and C are pyrimidines with 1 ring; also T has a CH 3 group on its ring.

III. DNA Replication How is genetic information replicated accurately at each cell division? Could each strand of the DNA double helix act as a template for the complementary strand? At each cell division, 10 9 base pairs are replicated. If error rate is 10 -6, then 3000 errors/cell division - TOO many.

DNA Replication is Semiconservative

Other Theoretical Possibilities

Separation of Nucleic Acids by CeCl Gradient Centrifugation

Meselson-Stahl Experiment DNA Labeling with 15 N Subsequent Generations Labeled with 14 N Cesium Chloride Gradient Banding

Expected Results From Conservative or Dispersive Reproduction If Conservative: Two bands, heavy and light, in 1st and 2nd generations If Dispersive, one smeary band in 1st and 2nd generations

Expected Results if Semiconservative These results were obtained. A related experiment was performed in plants (Fig. 12.5)

Bacterial DNA Replication begins at a Single Origin and Proceeds Bidirectionally Origin of Replication

DNA Polymerase I can Synthesize DNA Arthur Kornberg et al. (1957) discovered the enzyme in E. coli Requires template DNA strand, primer, MgCl2, and 4 dNTPs Monomers added 5’ to 3’

5’ to 3’ Addition of Monomers

DNA polymerases I, II and III pol I –most abundant (400/cell) –RNA primer removal pol II –unknown abundance –DNA repair? pol III –low abundance (15/cell) –DNA replication

Problems of DNA Synthesis Unwinding Tension must be relieved Priming Antiparallel strands RNA primer removal Backbone joining Proofreading

Steps of DNA Synthesis Denaturation and Unwinding Priming and Initiation Continuous and Discontinuous Synthesis –Including Proofreading and Error Correction Removal of Primer Ligation of nicks in backbone

Steps of DNA Synthesis: Denaturation and Unwinding of DNA DnaA, DnaB, DnaC proteins are helicases which bind origin and separate strands Single-strand binding protein (SSBP) keeps strands apart DNA gyrase, a type of DNA topoisomerase, cuts to relax supercoiling

Initiation of Synthesis RNA Primase makes RNA primer on DNA template DNA Polymerase III extends primer with DNA DNA Polymerase I removes RNA primer, replaces with DNA

Directionality of DNA synthesis

Proofreading occurs as polymerase moves along; if incorrect base pairing, base is removed and replaced.

Continuous and Discontinuous Synthesis Continuous on Leading Strand. Discontinuous on Lagging Strand creates Okazaki fragments. DNA ligase joins nicks in backbone.