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55-213 (36)
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Lecture 3

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Department
Biological Sciences
Course
55-213
Professor
Hubberstey
Semester
Winter

Description
Lecture 3 2013-01-24 4:25 AM Important%concepts%for%week%#2:% • Properties$of$nucleic$acids:$denaturation,$renaturation$and$hybridization$ o Properties$of$nucleic$acids$are$essential$to$understand$how$most$of$the$ inventions$and$discoveries$in$molecular$biology$were$done.$Many$of$them$ were$a$result$of$the$properties$of$nucleic$acids$that$allowed$molecular$ biologists$to$manipulate$them$and$study$them.$ • Mutation:$bad$or$good?$Why$is$DNA$mutation$important$for$survival?$ o Most$people$think$that$mutations$are$bad.$That’s$true$in$the$majority$cases.$ However,$in$multiple$examples,$they’re$good.$We$wouldn’t$be$here$today$if$it$ wasn’t$for$mutation.$It$is$essential$for$survival$and$evolution!$In$many$cases,$ it$has$allowed$many$species$to$survive$radical$environments.$We’ll$look$at$the$ natural$and$induced$causes$of$mutation.$ • [email protected]$genetic$inheritance;$importance$of$gene$copy$number$ o A$very$recent$discovery$from$6$years$ago.$Normally,$in$Mendelian$genetics,$ each$parent$contributes$a$set$of$genes:$1$maternal$copy$and$1$paternal$copy.$ Although$that$is$valid$for$the$majority$of$genes,$we$now$know$that$for$some$ genes$that’s$not$the$case;$you$don’t$get$1$copy$from$each$parent.$When$ people$were$compared$to$each$other,$molecular$biologists$found$there$were$ huge$gaps$in$our$genomes.$Some$people$may$be$carrying$more$or$less$than$ the$regular$2$copies$of$a$gene.$This$is$really$important$for$human$disease.$ This$variation$in$gene$copy$number$affects$human$evolution$and$disease.$$ $ Supercoiling%(Continued):% • See$last$lecture$slides$for$positive$and$negative$supercoiling$ • When$DNA$structure$was$discovered$by$Watson$and$Crick$in$the$1950s,$it$presented$ some$problems$in$our$understanding$of$how$processes$such$as$DNA$replication$and$ transcription$happened.$$ • If$you$twirl$an$elastic$and$try$to$separate$the$2$strands,$you’ll$have$a$problem.$This$ exactly$the$problem$that$happens$when$DNA$has$to$be$unwound$in$DNA$replication$ (copying)$and$transcription$(producing$complementary$RNA$strand).$$ • In$both$of$these$cases,$DNA$strand$has$to$separate$and$then$rejoin$at$the$end.$If$the$ cell$didn’t$compensate$for$this,$we$would$be$dead$as$replication$and$transcription$ would$be$inhibited.$$ • Thus,$DNA$has$to$separate$–$it’s$a$key$aspect$of$its$structure.$$ o See$figure$1.14$"$transcription$and$replication$require$DNA$strand$ separation$and$special$enzymes$ o See$figure$1.16$"$a$replication$fork$moves$along$DNA$ o See$figure$1.10$"$strand$separation$requires$changes$in$topology$ $ $ $ $ $ $ $ $ $ $ $ $ % % % $ $ $ $ $ Why%is%supercoiling%important?$ • DNA$strands$have$to$separate$during$replication,$transcription$and$recombination.$$ • Positive$supercoiling:$ o When$DNA$is$separated,$and$you$get$positive$supercoiling$occurring$at$one$ end,$how$does$the$cell$get$rid$of$it?$It$needs$to$get$rid$of$it$because$if$you$ keep$going,$eventually$the$strand$becomes$so$tight$that$the$enzymes$can’t$go$ down.$$ • How$does$this$torsional$stress$get$relieved$in$the$DNA?$The$only$physical$way$to$ detwist$an$elastic/DNA$is$to$cut$it$at$one$end$and$then$reseal$it.$That’s$when$the$ enzymes$topoisomerase$and$gyrase$do.$Thus,$enzymes$have$evolved$that$can$quickly$ detwist$the$strand.$ o A$nick$(aka$break)$is$generated$in$one$of$the$strands$which$allow$it$to$rotate$ to$relieve$tension.$ o Nick$is$resealed$(aka$ligated)$after$it$is$rotated$around$the$intact$strand$ • Many$proteins$(aka$enzymes)$are$involved$in$this$process$ o They$do$this$at$a$very$high$speed!$It$can’t$screw$up.$If$it$does,$mutations$will$ occur.$$ o This$process$occurs$every$second$of$your$life.$These$enzymes$do$this$during$ DNA$replication$or$transcription.$Remarkable!$ $ Types%of%Enzymes%Relieving%Torsional%Stress%in%DNA:% • Topoisomerases$ o Can$relax$OR$create$supercoils$in$DNA$ o Break$bonds$in$DNA$ ! Type$I:$single$strand$breaks$"$most$prevalent$type$ ! Type$II:$double$strand$breaks$ • Gyrases$ o Can$also$affect$supercoiling$ o Another$type$that$rotates$DNA$around$and$releases$some$of$the$supercoiling$ stress$ • They’re$primarily$involved$in$DNA$replication,$but$also$in$transcription.$ • This$shows$you$how$evolution$works;$evolution$has$kept$these$enzymes$because$ they$have$critical$functions.$$ • Our$cells$are$filled$with$other$enzymes$that$can$also$cut$DNA$or$nucleic$acid$in$ general$(see$below)$ $ Enzymes%Involved%in%DNA%Degradation:% • Deoxyribonucleases$(DNases)$degrade$DNA$ • Ribonucleases$(RNases)$degrade$RNA$ • Endonucleases:$break$bonds$within$nucleic$acid$ o See$figure$1.17$"$endonucleases$attack$internal$bonds$ $ o Which$endonucleases$are$used$in$molecular$biology?$ ! Restriction$enzymes$–$used$to$manipulate$DNA$in$vitro$in$the$lab$by$ cutting$DNA$at$specific$sequences.$Genetic$engineering$wouldn’t$be$ possible$without$them.$Where$are$they$generated$from?$Not$our$ cells,$but$bacteria$which$have$them$in$place$of$an$immune$system.$ Bacteria$can$be$infected$by$viruses;$these$restriction$enzymes$are$a$ defense$mechanism$which$can$take$the$foreign$DNA$of$the$virus$and$ cut$it$all$up,$hence$protecting$itself.$$ • Exonucleases:$break$bonds$at$ends$of$nucleic$acid$(5’$OR$3’$ends)$ o See$figure$1.18$"$exonucleases$nibble$from$the$ends$ o If$you$have$a$circular$molecule$of$DNA,$exonucleases$won’t$work.$They$can$ only$work$in$a$linear$piece$or$a$nicked$piece.$$ o Examples:$5’$exonuclease,$3’$exonuclease$ • These$enzymes$degrade$DNA/RNA$as$a$mechanism$to$protect$one$from$foreign$ DNA/RNA$coming$in.$Thus,$they’ve$evolved.$ • Molecular$biologists$have$taken$advantage$of$these$enzymes$by$isolating$them$and$ using$them$in$the$lab$to$cut$DNA$ $ DNA%Replication%is%SemiYConservative:$ • Back$when$DNA$structure$was$published$in$1950s,$it$wasn’t$sure$how$DNA$was$ replicated$in$the$cell$cycle.$Each$template$of$the$parental$DNA$strand$acts$as$a$ template$for$the$production$of$2$new$daughter$strands.$$ • Each$original$strand$of$the$DNA$duplex$serves$as$a$template$for$the$production$of$a$ new$strand$ 15 • Mesels[email protected]$Experiment$in$1958$labeled$DNA$with$“heavy”$isotope$of$N$( N)$and$ watched$generation$of$new$strands$ • Measured$the$weight$of$DNA$using$density$centrifugation$ • See$figure$1.15$"$DNA$single$strands$are$the$conserved$units$ • In$a$third$generation,$the$light$DNA$would$become$more$and$more$common$$ % % % % % % % % % % % % % % % % Nucleic%Acid%Hybridization:% • After$figuring$out$that$DNA$strands$could$come$apart,$molecular$biologists$used$that$ to$do$fancy$experiments$to$look$at$many$things.$$ • What$happens$if$you$isolate$2$DNA$from$different$organisms?$You$look$at$the$exact$ same$gene,$but$the$sequences$are$slightly$different.$The$hybridization$would$be$ affected$by$the$complementarity$of$the$2$sequences.$The$closer$aligned$2$species$ are,$the$more$similar$the$sequences$are,$and$the$higher$the$hybridization.$Not$only$ can$DNA$hybridize,$but$RNA$can$hybridize,[email protected]$combination!$ • All$nucleic$acids$can$hybridize$(form$H$bonds)$with$each$other$using$complementary$ sequences$ • [email protected]$"$how$are$genome$is$most$of$the$time,$this$is$the$structure.$But$ molecular$biologists$can$use$this$when$looking$at$the$DNA$of$2$different$organisms$ to$learn$something$about$the$sequence$of$that$species.$$ • [email protected]$ • [email protected]$ • Intra$or$intermolecular$bonds$ o In$the$intra$case:$if$you$have$a$single$RNA$strand$that$has$some$ complimentarity,$the$strand$can$flip$back,[email protected]$hybrid.$ This$can$happen$even$in$a$single$stranded$RNA$molecule.$$ o In$the$inter$case:$2$different$RNAs$come$and$combine$together$if$they$show$ complimentarity.$Could$also$ha
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