Microbio Kobryn Notes.docx

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Microbiology and Immunology
MCIM 326
Sydney Hayes

The Bacterial ChromosomeDNA is the molecule of heredityErwin Chargaff ratio GC and ATRosalind FranklinMaurice Wilkins Xray diffraction patterns of DNAdouble helixWatsonCrick published model of DNA structureNucleotidesDNA or RNA unit sugarbasePPurineA GPyrimidineT C UPhosphodiester bond between 3 and 5 of 2 different nucleotidesMinor Groove narrow less accessible bases less baserecognition by DNA binding proteins Major Groove wider more accessible more baserecognitionDouble helix of DNAHydrophobic basesHydrophilic sugarP backbone P is vely charged and repels each otherarrange to optimize P distanceBase stacking in centre of DNA hydrophobicHbonds via partially charged groups in bases establish AT GC basepairsantiparallel arrangement of strandsBasepairingStrand ComplementarityBase on one strand complementary to base on other strand Chargaffs ruleBasepairing allows DNA to replicate itself to make exact copies of each otherDNA Replication mostly same in all organisms on earthRibonucleotide reductase converts NDPs to dNDP RNA to DNA Hydroxyurea targetsKinase adds another P to make dNTP 2P to 3PdUTPPhosphatasedUMPthymidylate synthetasedTMPKinasedTTPThymidylate synthetase THFDHF dihydroxyfolate Dihydrofolate Reductase DHF THF tetrahydrofolateAntibiotic trimethoprim OR Anticancer drug methothrexate targets dihydrofolate reductase Trimethoprim kills cells by depletion of THF important for many biochemical pathways Thymidylate synthetase is the only enzyme in bacteria that converts THF to DHF DHF reductase is the only enzyme that restores THF but trimethoprim targets it Cells without thymidylate synthetase activity is resistant to trimethoprim ex import dTTPDeoxyribonucleotide PolymerizationDNA polymerases enzymes that join dNT together into polymers 2 exists in bacteriaPyrophosphate is a byproduct Phosphate of dNTP added to the 3 end OH of a second dNTPEnergy comes from the Pdiester bonds hydrolysisthe main energy currency in cellsPolymerase reads DNA 3 to 5DNA synthesized 5 to 3 by 3 end OH group attacking the next NTs P bondAntiparallel configuration after replication semiconservativeDNA polymerase can only extend preexisting DNA or RNA unlike RNA polymeraseDNA Polymerase III main replicative activity complex acting along many other factors at the replication forkDNA Polymerase I lagging strand replicationDNA repairNuclease breaks phosphodiester bonds in DNA and RNA degradation endo and exo5 end moves 5 to 3 and 3 endmoves 3 to 5 nucleasesPrimase makes short RNA primers that provide the 3end OH for DNA polymerase to synthsize DNALigase seals Pdiester bonds between Okazaki fragments on the lagging strand DNA joining short DNA chainsDNA Pol III Accesory ProteinsDNA Pol III Holoenzyme PolymerizationSliding clampClamp loading35 proof reading and exonucleaseaOrganizes complex joins leading and lagging DNA Pol III tethers helicase to Pol III holding all units togetherSliding Clampsubunit encircles DNA like a ring and preventssubunit from falling off template strandClamp Loadersubunits openclose sliding clamp SemiConservative Replication Each parental DNA strand is not destroyedMeselsonStahl Experiment 15Cells are grown in media containing heavy isotope of N then extracts DNACells are then moved to normal media with normal N for 2 more generations then extracts DNADNA with 2 heavy isotope strandheavy sink lower in centrifugation1 heavystrandmedium sink middle0 heavystrandlight sink less
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