Magnetic Traps– measuring Twist

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. polymers DNA DNA Structure • Right-hand helix • One turn: 3.4 nm, ~10.5 bp • Twist angle between bps θ=36 Molecular Cell Biology, Lodish Wikipedia 2nm 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. s Twist Writhe DNA may be Supercoiled:Has both Twist and Writhe Linking Number = Twist + Writhe Lk = Tw + Wr 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.

5. Some Examples of Tw and WrLinear DNA with Constrained Ends

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

7. Archaea are divided into four recognized phyla, but many more phyla may exist. 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. 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 3rd tree of life!different superhelicity Why? What about archebacteria that lives in hot springs? Positively supercoiled Makes DNA more stable

9. Low F: symmetric under s - s The shortening corresponds to the formation of plectonemes upon writhing. Torsionally stressed single DNA molecule Playing with phone cord: can you explain graphs? 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 s. 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. Extension vs. supercoiling at constant force Three regimes T. Strick et al., J. Stat. Phys., 93, 648-672, 1998

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

11. Nucleic Acids: DNA and RNA General Properties to Specific Properties DNA codes for genesGene: length of DNA which codes for a proteinCode 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)

12. Coding of DNA/RNA:3 bases are codon amino acid43= 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” Can look at genome: how many proteins are coded for? Answer: about 23,000 genes Count Start to Stop Codons: what fraction of 3 billion bases make up for proteins. 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. chromosome cell nucleus Double stranded DNA molecule Target Region for PCR DNA in the Cell How to identify you based on your DNA Individual nucleotides Polymerase Chain Reaction. Invented 1990; Nobel Prize in 1993: Kary Mullis

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

16. Original DNA target region Heat Heat Heat Cool Cool … PCR (Polymerase Chain Reaction) Copies DNA Exponentially through Multiple Thermal Cycles Oligo’s DNA Polymerase dNTP 1 copy 2 copies 4 copies In 32 cycles at 100% efficiency, ?? copies are created 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.

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.