DNA Replicaton 1 Flashcards
Describe Acyclovir and its importance.
It is a viral DNA polymerase inhibitor
> ThechemicalstructureofAcyclovirisguanineattachedtoan Incompleteribosering lackinga3’OHgroupnecessaryforpolymerization,thusactingasachainterminator.
What are 3 fundamental rules of DNA replication?
- It’s semiconservative
- It begins at an origin and proceeds bidirectionally
- Synthesis proceeds in a 5>3 direction and is semidiscontinous
What does it mean that DNA replication is semiconservative?
1.GeneticinformationistransmittedfromparenttoprogenybyreplicationofparentalDNA,aprocessinwhichtwodaughterDNAmoleculesareproducedthatareeachidenticaltotheparentalDNAmolecule.DuringDNAreplication,thetwocomplementarystrandsofparentalDNAarepulledapart.Eachoftheseparentalstrandsisthenusedasatemplateforthesynthesisofanewcomplementarystrand
(semiconservative replication)
2.Duringcelldivision,eachdaughtercellreceivesoneofthetwoidenticalDNAmolecules. 3.Eachdaughterduplexconsistsofoneparentalstrandandonenewlysynthesizedstrand 4.Comparedtoconservativereplicationwhereboth templateswouldgotothesamecell.
-figured out through radio labeling
Describe the origins of replication and which way they proceed?
Originsofreplicationareuniquesitesatwhichreplicationbegins.ThesesitesareATbasepairrichtofacilitatelocalmeltingoftheduplextossDNA necessaryforreplication
• WhyATbasepairs?
2.Replicationforksmoveinbothdirectionsawayfromtheorigin(“bidirectional”)formingareplicationbubblestructure.Asthetwostrandsunwindandseparatetheyformthe“V”shapedforkstructureswhereactivesynthesisoftheDNApolymeroccurs
3. Four sitesofDNAreplication
What is AAA+ ATPase?
- AAA+ (ATPases associatedwithdiversecellularactivities)ATPase– DnaA bindstotheoriginofreplicationanddisassociatesthehelicalstrands.TheenergyofATPcleavageisusedtoproduceaconformationalchangeinDnaA,whichforcesthestrandsapart
What does DNA ligase do?
Createsphosphodiester,bondsbyusingtheenergyofATPcleavage,thisseals“nicks”intheDNAstrand
What is the function of DNA Polymerase?
theseenzymesareresponsibleforstrandelongation,requirea
ssDNA asatemplate,andrequireanRNAprimer
What is the function of Helicase?
theseenzymescausedisassociationofthetwostrandsoftheDNAdoublehelix,unwindingthestructureusingtheenergyreleasedfromATPcleavage
What is the function of nucleases?
Nucleasesseverphosphodiester bondsoftheDNAbackbone.Theycanfunctionasanendonucleaseorexonuclease
What is Primase?
responsibleforsynthesizingshortstretchesofRNAcomplementarytothetemplateDNAstrandthatserveasaprimerforDNApolymerase
What is the function of Topoisomerases?
theseenzymesadjustthesupercoilingofDNAdoublehelices,bothalleviatingsupercoilingstressandintroducingnegativesupercoiling.Theycontainbothendonucleasefunctionandligasefuction.TypeItopoisomerasescleaveoneofthestrandsofthedoublehelix,TypeIITopoisomerasescleavebothstrandstoperformtheirfunctions
Describe the process of DNA replication initiation.
- DnaA binds to the site of replication.
- DNA Helicase dissociates double helix
- Single strand DNA binding protein keeps two strands of DNA separate.
Describe the process of elongation, leading and lagging strand synthesis.
DNApolymeraseIIIbeginssynthesizingDNAinthe5’to 3’direction,beginningatthe3’endofeachRNAprimer.Thenewlysynthesizedstrandiscomplementaryandantiparalleltotheparentalstrandusedasatemplate.Thisstrandcanbemadecontinuouslyinonelongpieceandisknownasthe“leadingstrand”
- The“laggingstrand”issynthesizeddiscontinuouslyasaseriesofsmallfragments(about1000nucleotideslong)knownasOkazakifragments.EachOkazakifragmentisinitiatedbythesynthesisofanRNAprimerbyprimase,andthencompletedbythesynthesisofDNAbyDNApolymeraseIII.Eachfragmentismadeinthe5’to 3’direction
- dNTP todNMP and2Pidrivessynthesis 4.RNAprimersareremoved
- accompanied by proofreading functions and Topoisomerases crucial for alleviating supercoiling
What is the difference between the two types of Topoisomerases?
- Topoisomerases cut double helix to allow stress to alleviate
- Type I cuts one strand
- Type II (DNA gyrase) cuts two stands, it alleviates positive super coiling between imposed by stand unwinding
What are some mechanisms for Topoisomerases II inhibition and what are they used for?
1.AlleviationofDNAsupercoilingbytopoisomerasesandareimportantdrugtargets.AlltopoisomeraseII-directedagentsareabletointerferewithatleastonestepofthecatalyticcycle.AgentsabletostabilizethecovalentDNAtopoisomeraseIIcomplex(alsoknownasthecleavablecomplex)aretraditionallycalledtopoisomeraseIIpoisons,whileagentsactingonanyoftheotherstepsinthecatalyticcyclearecalledcatalyticinhibitors.WhereastopoisomeraseIIpoisonsareusedsolelyfortheirantitumoractivities,catalytic
inhibitorsareutilizedforavarietyofreasons,includingtheiractivityas
antineoplasticagents(aclarubicin),cardioprotectors (ICRF-187),ormodulatorsinordertoincreasetheefficacyofotheragents(suramin andnovobiocin).
How does DNA proofreading work?
When DNA polymerase mispairs a nucleotide with the template it uses 3’ > 5’ exonuclease activity to excise the mismatched nucleotide
Describe DNA replication termination.
- Occurs when the replication forks join up
- Specific sequences are involved and there are proteins to resolve the two newly formed chromosomes
- Involves DNAgyrase,whichisatopoisomerasetypeII
What are three things that insure genome fidelity?
- Geometry of active site of DNA polymerase
- Proofreading - 3’ > 5’ exonuclease
- Mismatch repair and DNA methylation
-estimated that less than one mutation introduced per cell division
Describe the different prokaryotic DNA polymerases.
1.Despiteitsearlycharacterization,itquicklybecameapparentthatPolymeraseIwasnottheenzymeresponsibleformostDNAsynthesis— DNAreplicationinE.coliproceeds atapproximately1,000nucleotides/second,whiletherateofbasepairsynthesisbyPolymeraseIaveragesonlybetween10and20nucleotides/second.Moreover,itscellularabundanceofapproximately400moleculespercelldidnotcorrelatewiththefactthattherearetypicallyonlytworeplicationforksinE.coli.Moreover,itisinsufficientlyprocessive tocopyanentiregenome,asitfallsoffafterincorporatingonly
25‐50nucleotides.
2. Processivity ofDNAPolymeraseIII
3. DNApolymeraseIhasaspecial5’‐3’exonuclease activity
4.BothDNApolymeraseIandIIIhaveproofreadingfunctionthatisanaspectof3’ > 5’exonuclease activity.
- I (DNA repair and removing RNA primers), II-IV-V (DNA repair functions), III (synth most DNA of leading and lagging strands, has ability to latch on and stay)