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Announcements1. 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 2.
4. 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 20. 15 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, GC
• 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
GC
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 GC C A A C C C
T T T G C G CA AA C G C G
1 2
3
U U U G C G C
Transcription of 1 strandof 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 G3
21
Heat - denature
U U U G C G CA 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 GC C A A C C C
Nucleic Acid Hybridization
Nucleic Acid Gel Electrophoresis
Base Pairing RulesWhat makesnucleic acidsacidic?
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 CH3 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, 109 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 15N
Subsequent Generations Labeled with 14N
Cesium Chloride Gradient Banding
Expected Results From Conservative or Dispersive Reproduction
If Conservative: Twobands, heavy and light,in 1st and 2nd generations
If Dispersive, one smearyband in 1st and 2ndgenerations
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 ofReplication
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
Proofreadingoccurs as polymerasemoves along; ifincorrect base pairing, base is removed and replaced.
Continuous and Discontinuous Synthesis
• Continuouson Leading Strand. • Discontinuouson Lagging Strandcreates Okazakifragments.
• DNA ligase joinsnicks in backbone.