Exam 2 Study GuideLECTURE 1: SUPERCOILING/CHROMOSOME STRUCTUREElectron micrographLoopsE.Coli is a single, covalently closed circular DNA (20 Angstroms, 4.6 billion basepairs)DNA – 1 mm long, cell is 1 micron (1000 times smaller) long SupercoilingMovement in electrophoresis depends on:ChargeShapeALL CELLS- compaction of DNANicked Circle – one thick bandLinear – one thick, one thinSupercoilied – two even bandsEach one of our cells has to have PACKED DNA, because we have 1 meter of DNA and we put it into a cell we can’t even see!How?DNA is supercoiled***B DNA – 10.5 base pairs/turnMost stableNatural folding (energetically favorable) under physiological conditionsCCCDNA - Two ends of DNA attached to one anotherEx: Plasmid or entire E.Coli chromosomeToplogically constrained (attached to itself)Cells regulate topological constraintClosed Circle (coiled)Linear (relaxed)NEGATIVELY SUPERCOILED – superhelix is layered onto normal DNA helix (much more compact than normal DNA situations)Supercoiling does NOT mean that DNA is NOT B-form DNA. Coiling superimposed ON B-form DNA.Most DNA is about 5% underwound. 10.5 kb relaxed has about 1000 turns, CCC DNA from cell has about 950 (less turns)MATH OF SUPERCOILING (Don’t go anywhere without your hose)Covalently closed DNA has linking number - # of times the DNA is would around somethingDiffers from the next band by 1 when further from wellsIn order to change the linking #, you have to BREAK a covalent bond (otherwise it WON’T CHANGE)1. TWIST – number of helical turns2. WRITHE – number of times the helix crosses itselfa. LK = TW + WREX: # Base Pairs/10 base pairs per turn = 36 LK (DNA is would 36 times)BFORM IS ALWAYS 10 BP per turnLK up or down? Breaking a covalent bondUnwinding by 4 turns – what happens to DNA?DNA want to stay B-form (keeps 10 bp/turn – En Stable)Twist will be unchangedWRITHEs in space by 4. The Helix crosses itself by 4. (Negatively supercoiling) (VISUALIZATION*** like twisting a circular piece of streamer)PARTIAL unwinding – crucial for functions in cell.Tries to wind back because of most energetically favorable form of DNA.OVERwind – wring it harder/tighterUNDERwind – let it looseEX: Two turns unwound = un coiled = underwound = lower linking number by 4 (4 turns).(bonds broken)TW and WR are interconvertible.*NOTHING SPECIAL about supercoiled DNA, it’s the exact same as normal DNA just changed in shape (NOT STRUCTURE)*Assume with DNA with 10bp/helical turn has TW=36.DNA is underwound often because we need to gain ACCESS to the DNA bases. Have to literally UNWIND DNA for DNA transcription initiation. (ORIC)Twist decreases (unwound certain # of turns, writhe GOES AWAY)Topologically equivalent if LINKING NUMBER remains same.WRITHE compensates for loss in # of turns (LK)In the cell, the DNA is MAINTAINED IN NEGATIVE SUPERCOIL.Where POSITIVE supercoiling??Moving replication fork through DNA = unwinding DNA(VISUALIZATION*** Rubber band folded in half twisted together (DNA) and one side begins to separate strands  other side becomes more and more wound, turns get pushed forward with less distance in between  This is POSITIVE overwound supercoiling) Replication Fork, writhes in space to COMPENSATE.ENZYMES in place to relax again to counteract the positiveWithout these counteracting enzymes, we would not be able to replicate DNA with a replication fork.Negative supercoiling is spontaneous (similar to pattern of a telephone cord, writhing in space)More compactFound in cellsTopologically equivalentEnzymes called TOPOISOMERASES (Isomerizing the topological states of helical structures of DNA)Break bondsPivot after a nick is madeSingle strand breaks  Energy builds up into negatively supercoiled DNA.DNA spins/rotates and relaxes until back to B-Form DNA. (releasing energy  low energy state  relaxed state) Process of Relaxing DNA.TYPES OF TOPOISOMERASESType II TopoisomeraseE. Coli – Topo II and Topo IVHumans – Topo II and Topo IVENERGY REQUIRED (ATP)Pass/cut both pieces of DNA and pass another piece throughTOPO II binds: Covalent intermediate 5’phosphate  Tyrosine (AA)All Topoisomerase II’s act to relax supercoiled DNA, or remove supercoils EXCEPT for Gyrase.Cuts through overlap of strands, top strand first. Goes back and puts strand over top strand. And reseals break with opposite layer on top.GYRASEAdds negative supercoils (coils further)Changes LK by -2 each time it acts***only in prokaryotes (bacteria, E. Coli)target of quinolone antibiotics (helps with parkinson’s, humans don’t have gyrase)Gyrase (multisubunit) binds to DNA, wrapped around complex in N-gate making + node, 5’ linkages are made after DNA is cleaved, strand passes through and exits enzyme through C-gate. Then strands are religated.ATP is used for conformational change to move piece of DNA to other side, not for making the break.Type I TopoisomerasesE. Coli Topo I and Topo IIIRelax DNA, make fewer supercoilsDon’t require ATPChange linking # by +1.Covalent intermediate – 5’ phosphotyrosine linkage to the enzyme.Pass ONE strand through the other. (nick, pass strand through break, and ligate)LK = n + 1Smaller scale: not passing double stranded pair through (Type II). Only passing one strand of the double strand through.Energy is inherent in superhelicity of the DNA.Energy is maintained in phosphodiester bond. (can be reversed when ligated with normal bond)Covalent intermediate maintains energy between 3’-hydroxyl and 5’-phosphotyrosylCommon reaction in DNA-modifying enzymes**GEL ELECTROPHORESISDependent on CHARGE and SHAPE.Diff topological states = diff shapes = move differentlyNicked Circle, Linear, Supercoiled = can all be same DNA in different topological statesNicked circle moves slowest than linear and compact moves furthest.Relaxed DNA moves least, highly supercoiled moves furthest. (less LK) Why?Mixture of DNA is treated with Type I topoisomerase. Grabs a hold and relaxes by 1 over and over again so we can see a range of bands differing by 1 LK.What do TOPOISOMERASES do??1. Relax supercoiled DNA (except gyrase that adds negative supercoils)2. Untangle Knots3. Resolve (decatenate) catenanes (rings stuck in each other *** look like Olympic rings)ALL cells require topoisomerases.Some are specialized in those three tasks.E. Coli has 4, Mammals have 6 (5 essential)ALL Topo’s besides Gyrase can only relax DNA. Using energy inherent in molecule.GYRASE = -2 (turns further)Catenation = strands