1. Magnetic Traps– measuring Twist Last time: WLC very good theory for DNA bending This time: Twist & Writhe General Properties of DNA to Specific: PCR HW due next Monday; also quiz #1 on Chpt 1 of Phillips et al

2. DNA Structure Molecular Cell Biology, Lodish Wikipedia Right-hand helix One turn: 3.4 nm, ~10.5 bp Twist angle between bps θ=36 2nm polymers DNA DNA will resist twisting

3. DNA in Eukaryotes is wrapped around Histones (Proteins) Chromatin = Complex of DNA + Protein (histones + non-histones) 8/17/06

4. DNA may be Supercoiled: Has both Twist and Writhe  Twist Writhe Relaxed DNA: Twist =1 turn/10.4 bp. Writhe = 0. ∆Tw=0 Can’t just twist up DNA and have it all go into twist. Example: Phone cord. Pull on DNA and writhe comes out. Measure relaxed (non super-coiled) DNA and figure out length vs. force. Linking Number = Twist + Writhe Lk = Tw + Wr

5. Some Examples of Tw and Wr Linear DNA with Constrained Ends

6. Twist (T w ), Writhe (W r ), Linking Number (Lk) T w : # of times the two strands wrap around each other W r : # of times C crosses itself. Charvin, Contemporary Physics,2004 T w =2 T w =0 W r =0 W r =2 Linking Number = Twist + Writhe Lk = Tw + Wr Ex: Hold DNA out straight so that it has no Writhe, add of take out twist, then let fold up (Twist goes into Writhe).  = (Lk – Lk o )/Lk o =∆Lk/ Lk o Supercoiled DNA (  ): is the deviation from relaxed linking number. Normal DNA is negatively supercoiled, -0.06 = 6 turns for every 100 taken out. Why? Positively supercoiled Helps unwind DNA– makes it easier to uncoil, separate strands. Enzymes which do this called Topoisomerases. Makes DNA more stable Why? What about archebacteria that lives in hot springs?

7. Archea—live hot! The Archaea are a group of single-celled microorganisms. A single individual or species from this domain is called an archaeon (sometimes spelled "archeon"). They have no cell nucleus or any other membrane- bound organelles within their cells. Archaea are divided into four recognized phyla, but many more phyla may exist. Finding Archaea : The hot springs of Yellowstone National Park, USA, were among the first places Archaea were discovered. At left is Octopus Spring, and at right is Grand Prismatic Pool. Each pool has slightly different mineral content, temperature, salinity, etc., so different pools may contain different communities of archaeans and other microbes. The biologists pictured above are immersing microscope slides in the boiling pool onto which some archaeans might be captured for study. http://www.ucmp.berkeley.edu/archaea/archaea.html

8. Archaea— the 3 rd tree of life! different superhelicity Why? What about archebacteria that lives in hot springs? Positively supercoiled Makes DNA more stable

9. Torsionally stressed single DNA molecule T. Strick et al., J. Stat. Phys., 93, 648-672, 1998 Extension vs. supercoiling at constant force Three regimes Low F: symmetric under   -  The shortening corresponds to the formation of plectonemes upon writhing. When the force is increased above 0.5 pN, the curve becomes asymmetric: supercoils still form for positive coiling while local denaturation adsorbs the torsional stress for negative . At forces larger than 3 pN no plectonemes are observed: the torsional stress is adsorbed not by writhe but in local structural changes of the molecule. Playing with phone cord: can you explain graphs?

10. Over- and under-stretching Rotation extension curves for different forces. At higher forces one cannot induce supercoils but denature the DNA molecule. Upon twisting a DNA molecule it takes a number of turns, before the DNA length reduces significantly and plectonemes are formed. The point (N buckling ) where DNA starts to form plectonemes with a constant length reduction per turn is called buckling instability

11. DNA codes for genes Gene: length of DNA which codes for a protein Code first goes through RNA. RNA: every 3 bases codes for amino acid. Peptide = linear string of amino acids (from 10kD (≈ 100aa) to 1 MD or more) Nucleic Acids: DNA and RNA General Properties to Specific Properties

12. Coding of DNA/RNA: 3 bases are codon  amino acid 4 3 = 64 possible amino acids, but there are only 20 aa. Degeneracy aa = AT1, AT2, AT3… Plus stop and start codons (where proteins stop, start). All genes start with ATG  proteins start with Methionine All Genes stop with: TAG: "They Are Gone" TAA: "They Are Away" TGA: "They're Going Away” Count Start to Stop Codons: what fraction of 3 billion bases make up for proteins. Can look at genome: how many proteins are coded for? Answer: about 23,000 genes Only about 10% is used for genes. 90% used to be called “junk” DNA.

13. “Junk” DNA codes for important regulatory elements Produce RNA, RNA then feeds back onto DNA and turns it on/off in some unclear manner. “Rethinking ‘Junk’ DNA” NYTimes 9/5/12 “Bits of Mystery DNA, Far From ‘Junk,’ Play Crucial Role” Cancer is one result. “Cancer’s Secrets Come Into Sharper Focus: NYTimes Some junk DNA is truly “junk”—evolutionary baggage.

14. DNA in the Cell chromosome cell nucleus Double stranded DNA molecule Individual nucleotides Polymerase Chain Reaction. Invented 1990; Nobel Prize in 1993: Kary Mullis Target Region for PCR How to identify you based on your DNA

15. Make copies (extend primers) Starting DNA Template 5’5’ 5’5’ 3’3’ 3’3’ 5’5’ 5’5’ 3’3’ 3’3’ Add primers (anneal) 5’5’ 3’3’ 3’3’ 5’5’ Forward primer Reverse primer DNA Amplification with the Polymerase Chain Reaction (PCR) Separate strands (denature) 5’5’ 5’5’ 3’3’ 3’3’

16. In 32 cycles at 100% efficiency, ?? copies are created PCR (Polymerase Chain Reaction) Copies DNA Exponentially through Multiple Thermal Cycles Original DNA target region Heat Cool Heat DNA Polymerase dNTP Oligo’s 1 copy 2 copies Cool 4 copies Heat … 1.07 billion To work, what property of DNA polymerase has to have? Heat stable so don’t have to add in new polymerase for every cycle Thermostable organisms, e.g. living in Yellowstone Geysers have this. New Scientists (1998)…Yellowstone's bugs land up in court... Microorganisms from hot springs are especially valuable because their enzymes are not easily destroyed by heat....

17. Applications of PCR (next time)

18. Class evaluation 1. What was the most interesting thing you learned in class today? 2. What are you confused about? 3. Related to today’s subject, what would you like to know more about? 4. Any helpful comments. Answer, and turn in at the end of class.