MCB 502A-2015. Lecture # 9. Topoisomerases. The replisome.— DNA gyrase— Type I and type II topoisomerases— Bacterial NucleoidReplication factory— The composition of DNA polymerase III— The structure and function of the replication fork in E. coli. — The replisome that moves in opposite directions at the same time— The replication factoryDNA gyrase-1— The second topoisomerase… very different …. —… opposite reaction, … highly negatively-supercoiled one. — … required ATP… (without ATP, … relax negatively-supercoiled DNA… ). — … in steps of two… . ATP ATPPositive scNegative scNo ATPΔL# = 2Marty GellertGyrBGyrBDNA gyrase-2— … heterotetramer … — … "Topo II", … DNA gyrase, ….— The elucidation of the mechanism … three characteristics …: 1) the two subunits can be isolated separately … ; GyrAGyrBGyrBGyrAGyrA GyrAGyrBGyrBGyrA GyrAGyrBGyrBDNA gyrase-32) … specific antibiotic …;3) mutations in gyr genes… — … mix-and-match exercise. — Whenever one of the two inhibitors blocked a particular reaction, … :Sensitivity to antibioticProperty NAL COUNegative supercoiling — —DNA-dependent ATPase ± —Relaxation without ATP — +Linearization with SDS — +Subunit assignment GyrA GyrB Function Catalysis ATPase Nalidixic acidCoumermycinGyrA GyrADNA gyrase-4— Negative supercoiling of circular DNA … — ATP hydrolysis in the presence of dsDNA is blocked by COU, … — … ATP-hydrolysis is a function of GyrB. — Relaxation of negatively supercoiled DNA… … catalyzed by GyrA. Sensitivity to antibioticProperty NAL COUNegative supercoiling — —DNA-dependent ATPase ± —Relaxation without ATP — +Linearization with SDS — +Subunit assignment GyrA GyrB Function Catalysis ATPase — .. the emerging picture …DNA gyrase-5— What kind of action? — … adding SDS … NAL-inhibited gyrase reaction… — … staggered 4 bp 5’-protruding ends, … via phospho-tyrosyl bond. — … linearization is not inhibited by COU…… not observed in complete absence of ATP, … GyrAGyrAGyraseSDSDNA gyrase-6— … new: in contrast to topo I … DSB … passes another double-strand segment … — … explains … in steps of two. Upper gateMain gateLower gateUpper openAll lockedMain openLower openAll locked1234Individual stages explained1) Catch the ”blue" duplex.2) Break the ”navy" duplex, pass the ”blue" duplex through the double-strand opening.3) Seal the break in the navy duplex, release the blue duplex.4) Return to the original "ready-to-catch" configuration.DNA gyrase-7— … which way to go … ? — … directionality … the relaxation reaction by Topo I…. — … unclear how gyrase … — … extensive contacts with the substrate DNA … manipulates two DNA segments …DNA gyrase-8— … a protein binding a particular site on DNA… … the "nuclease protection" characterization… — … only … site-preference, … — … DNA gyrase …. Bacteriophage Mu chromosome … Phage MuControlNo proteinBound proteinprotected"Very limited" nuclease treatment delivers on average only one cut per DNA molecule.Gyrase on its strong binding site in phage Mu chromosomehttp://jb.asm.org/content/188/2/619.fullhttp://viralzone.expasy.org/all_by_species/4356.htmlDNA gyrase-8— … DNA gyrase occupies around 150 bp of DNA… (374 kDa) … DNA wrapping around gyraseDNA gyrase-9— … bound to a nicked circle, …. sealed with DNA ligase… positive supercoiling …. — … not blocked by either NAL or COU, … precedes the breakage-reunion cycle … property … binding to DNA. — … part … passive induction of positive supercoiling … subsequent enzyme-catalyzed compensatory "relaxation" … (+)sc!DNA gyraseNo ATPDNA ligaseNADHPhenol to deproteiniseDNA gyrase-10— … model …wraps a DNA loop … breaks one part of this loop …— … the two DNA segments … same DNA molecule … — … substrate of DNA gyrase in vivo.DNA gyrase-11— An exception that proves the rule … … catenation / decatenation … can hold two non-contiguous DNA sequences. — … 100 times less efficiently than negative supercoiling of a single DNA molecule… catenaneshttp://www.biochem.arizona.edu/classes/bioc461/Chapter27Notes.htmType I and type II topoisomerases-1— … two more topoisomerases … E. coli: Topo III,… and Topo IV, … — The function of Topo III is still unknown …— Topo IV is the decatenase of the E. coli cells …. . …also a major relaxase … (together with Topo I).Topo IVType I and type II topoisomerases-2— Many more … all three kingdoms …. — .. the linking number in steps of 3 or 4. — … subdivided into two classes: type I enzymes … type II enzymes …The three general functions of topoisomareseshttp://open.jorum.ac.uk/xmlui/bitstream/handle/10949/956/Items/S377_1_section11.html?sequence=12Type I and type II topoisomerases-3— … why are there two classes instead of … one? — … the cell needs both types of generic enzymes: … — … site-specific recombinases, — are close relatives of type I topoisomerases. — …"decatenases" — are Type II topoisomerases. — Topo I "relaxase" and DNA gyrase …Type I and type II topoisomerases-4— The biological functions of the two topoisomerase types… — Type II topoisomerases : 1) … maintaining negative supercoiling …; 2) decatenation of sister DNA… — Type I topo: … introducing missing coiling … fine-tuning …. http://www.nature.com/nrc/journal/v6/n10/fig_tab/nrc1977_F1.htmlhttp://www.nature.com/nrd/journal/v6/n1/fig_tab/nrd2202_F3.htmlType I and type II topoisomerases-5— … gyrase … replication fork progress. — Addition of 4-quinolone derivatives (nalidixic acid, ciprofloxacin) …. — Importantly, 4-quinolones … do not have targets in eukaryotic cells. — … (CIPRO) … against a broad range of eubacterial infections.TCA-precipitable 3H-dT (DNA)Time (hours)0 1 2noNalidixic acidRifampicinTreatmentAddition of rifampicin mimics the phenotype of delayed-stop dna mutantsAddition of Nalidixic acid mimics the phenotype of immediate-stop dna mutantsType I and type II topoisomerases-6— … why the replication fork progress … type II topo … rather than a type I …? — … DNA unwinding by DNA helicases at the replication fork … … maintained by DNA gyrase. — … topo I enzymes of bacteria do not relax
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