Fig 4-0Topological Linking Number (Lk): Twist (Tw) + Writhe (Wr)Tw = “the number of times Watson strand crosses Crick”(the number of turns of B-form DNA: right-handed turns = +)Wr = “the number of times WC duplex crosses WC duplex”(supercoiling: compaction of B-form DNA against itself;right handed supercoils are +, left handed supercoils are -)Lk = 6Tw = 6Wr = 0Lko = most favorable Lk =B-form DNA with no supercoilsFig 4-1DNA in cells is negatively supercoiled. Twist andwrithe can interconvert, but linking number cannotwithout breaking the phosphodiester backbone.Fig 4-2negative supercoil(‘left-handed’ writhe)positive supercoil(‘right-handed’ writhe)Tw = 6Wr = 0Tw = 6Wr = -1 +1Lk = 6no enzymeis requiredTw = 6Wr = -1Lk = 5enzyme isrequired!Lk = 6must remove + supercoilsnucleosomeDNA packaging introduces a topological problem“topoisomers” differ in Lk; Lk is altered by “topoisomerase” enzymesFig 4-3Changes Lk in steps of 2.Enzyme breaks one duplex and passes another duplex through the break.Type II topoisomerases can bias change in Lk in any direction using ATPhydrolysis to drive a conformational change in the enzyme.Two kinds of topoisomerasesType I: cuts one strandType II: cuts two strandsBOTH types form a covalent protein-DNA intermediate at astrand break to store phosphodiester bond energy. Unlike restrictionenzymes, cleavage is reversible and strands join without DNA ligase.Changes Lk in steps of 1.Enzyme nicks one strand and rotates it around the other strand.Type I topoisomerases can act to change Lk only towards Lko.Fig 4-4Type I Topoisomerase(for example, E. coli topo I)Fig 4-5A topoisomerase II dimer makes a DNA breakTyrosine OHattacks PO4 andforms a covalentintermediateStructuralchanges in theprotein open thegap by 20 Å!Fig 4-6E. coli gyrase changes Lk in steps of -2: itbinds to a + supercoil and converts it to -supercoil.Type II Topoisomerase (for example, E. coli DNA gyrase)Gyrase is the target of several anti-bacterial drugsFig 4-7A type II topoisomerase candecatenate linked dsDNAsFig 4-8DNA topology changes and needs to bechanged on a continuous basis(+) supercoils(-) supercoils(+) supercoilsFig 4-9Once genome replication starts, it should finish(otherwise some genes are amplified).As a result, the initiation of DNA replication is highly regulated.Initiation requires building replication forks.Fig 4-10electron micrographof replicatingDNA circleone possibleinterpretationof events (thecorrect one)Fig 4-11If replicating cells are exposed to a pulse of[3H]thymidine, the resulting DNA will be heavily labeled(“hot”) where early replication occurs and lightly labeledfarther away. When such labeled DNA is dried on amicroscope slide as long fibers and exposed to a radiation-sensitive emulsion, autoradiographic signals should beproduced proportional to the hot-ness of the DNA.ORI = an origin of replicationBidirectional movement of replication machinery from the site of replication initiationFig 4-12To have bidirectional replication from an origin, leading strand synthesis must be started twice.The origin “firing” event needs toresult in loading of two DnaB hexamers, one on each strand.Fig 4-13Finding E. coli oriCFig 4-14A DNA fragment of interest (or apool of fragments) is ligated into aautonomously replicating vector,then introduced into a bacterial host.It can be replicated indefinitely.Plasmids are extrachromosomal andcan be easily purified away from themuch larger host cell chromosometo isolate the cloned sequence.Ligation of a pool of insertfragments to the vector moleculescreates a library of clones.A replication originand resistance markerallow DNA cloningFig 4-15 A typical cloning vector for E. coliOrigin + selectable marker + DNA insertion site (polylinker of many unique restriction sites)Fig 4-16E. coli oriC : the origin of DNA replicationThe oriC sequence is necessary and sufficient for replication of a circular DNA in E. coli,but the DNA will replicate only once per cell division cycle (like the chromosome).Plasmids with oriC have low copy number. Most plasmids used for cloning have a phage origin and high copy number to get more DNA.13 bp repeats 9 bp repeatsBinding sites for DnaA proteinAT-richFig 4-17• ATP binding increases DnaA affinity for DNA• many (~30) DnaA monomers bind cooperatively to 9 bp repeats and flanking DNA• Wrapping of DNA around DnaA favors the unpairing ofadjacent AT-rich sequence• DnaA loads DnaB onto each strand • DnaB hexamer can’t get on DNA without opening the hexameric ring; a DnaC hexamer loads DnaB• DNA binding by DnaB releases DnaC• DnaA hydrolysis of ATP promotes disassemblyE. coli DnaA protein controls origin firing for replicationFig 4-18Crystal structure of DnaA reveals the mechanism of DNA wrappingThe arrangement of DNA binding sites introduces positive(right handed) supercoils by wrapping DNA on the outside.Compensating strand unpairing in the adjacent AT-rich regioncreates a replication bubble reading for loading of DnaB. Fig 4-191 L culture = 4.1010 cells --> 400 000 km DNA synthesized (Earth-Moon distance)Yeast14 Mbp 3 kb/min 20 min ~330S would last 80 hr if 1 ori2.1013 km DNA synthesized (2 light-years) during life time (1016 cell divisions)Human3 Gbp 3 kb/min 7 h >10 000 ?S would last 1 year if 1 oriGenome Fork speed S phase Origins CommentE. coli4.6 Mbp30 kb/min 40 min 1Eukaryotes need multiple replication originsEukaryotes need multiple replication originsS phase = DNA SynthesisFig 4-20... but not all yeast ARS (autonomouslyreplicating sequences)are used as origins ... and same experimentdoesn’t find any humanDNA pieces able to serve as originsIdentification of eukaryotic replication originsFig 4-21G1: inactive but readyS: activeG2, M: inactiveEukaryotic DNA replication originsare regulated with the cell cycle:enabled in G1, activated in S, and deactivated by DNA replication.Phases of the cell cycle:G1 growthS genome replicationG2 growthM cell divisionFig 4-22Eukaryotic factors for initiation of DNA replicationModel systems: invading genomes Bacteriophage lambda lO binds origin, lP allows loading of DnaBSV-40 virus T antigen binds the origin AND is the helicase• Origin recognition: Origin Recognition Complex (ORC)• DNA helicase: MCM2-7 (heterohexamer)• Many other proteins interact with ORC and MCM to regulate the sites and timing of replication initiation.to be infective these must overcome host regulationof chromosome replication once per cell cycleFig