DNA Replicaton 1

Question 1

Describe Acyclovir and its importance.

Answer 1

It is a viral DNA polymerase inhibitor

> ThechemicalstructureofAcyclovirisguanineattachedtoan Incompleteribosering lackinga3’OHgroupnecessaryforpolymerization,thusactingasachainterminator.

Question 2

What are 3 fundamental rules of DNA replication?

Answer 2

1. It’s semiconservative
2. It begins at an origin and proceeds bidirectionally
3. Synthesis proceeds in a 5>3 direction and is semidiscontinous

Question 3

What does it mean that DNA replication is semiconservative?

Answer 3

1.GeneticinformationistransmittedfromparenttoprogenybyreplicationofparentalDNA,aprocessinwhichtwodaughterDNAmoleculesareproducedthatareeachidenticaltotheparentalDNAmolecule.DuringDNAreplication,thetwocomplementarystrandsofparentalDNAarepulledapart.Eachoftheseparentalstrandsisthenusedasatemplateforthesynthesisofanewcomplementarystrand
(semiconservative replication)
2.Duringcelldivision,eachdaughtercellreceivesoneofthetwoidenticalDNAmolecules. 3.Eachdaughterduplexconsistsofoneparentalstrandandonenewlysynthesizedstrand 4.Comparedtoconservativereplicationwhereboth templateswouldgotothesamecell.
-figured out through radio labeling

Question 4

Describe the origins of replication and which way they proceed?

Answer 4

Originsofreplicationareuniquesitesatwhichreplicationbegins.ThesesitesareATbasepairrichtofacilitatelocalmeltingoftheduplextossDNA necessaryforreplication
• WhyATbasepairs?
2.Replicationforksmoveinbothdirectionsawayfromtheorigin(“bidirectional”)formingareplicationbubblestructure.Asthetwostrandsunwindandseparatetheyformthe“V”shapedforkstructureswhereactivesynthesisoftheDNApolymeroccurs
3. Four sitesofDNAreplication

Question 5

What is AAA+ ATPase?

Answer 5

1. AAA+ (ATPases associatedwithdiversecellularactivities)ATPase– DnaA bindstotheoriginofreplicationanddisassociatesthehelicalstrands.TheenergyofATPcleavageisusedtoproduceaconformationalchangeinDnaA,whichforcesthestrandsapart

Question 6

What does DNA ligase do?

Answer 6

Createsphosphodiester,bondsbyusingtheenergyofATPcleavage,thisseals“nicks”intheDNAstrand

Question 7

What is the function of DNA Polymerase?

Answer 7

theseenzymesareresponsibleforstrandelongation,requirea
ssDNA asatemplate,andrequireanRNAprimer

Question 8

What is the function of Helicase?

Answer 8

theseenzymescausedisassociationofthetwostrandsoftheDNAdoublehelix,unwindingthestructureusingtheenergyreleasedfromATPcleavage

Question 9

What is the function of nucleases?

Answer 9

Nucleasesseverphosphodiester bondsoftheDNAbackbone.Theycanfunctionasanendonucleaseorexonuclease

Question 10

What is Primase?

Answer 10

responsibleforsynthesizingshortstretchesofRNAcomplementarytothetemplateDNAstrandthatserveasaprimerforDNApolymerase

Question 11

What is the function of Topoisomerases?

Answer 11

theseenzymesadjustthesupercoilingofDNAdoublehelices,bothalleviatingsupercoilingstressandintroducingnegativesupercoiling.Theycontainbothendonucleasefunctionandligasefuction.TypeItopoisomerasescleaveoneofthestrandsofthedoublehelix,TypeIITopoisomerasescleavebothstrandstoperformtheirfunctions

Question 12

Describe the process of DNA replication initiation.

Answer 12

• DnaA binds to the site of replication.
• DNA Helicase dissociates double helix
• Single strand DNA binding protein keeps two strands of DNA separate.

Question 13

Describe the process of elongation, leading and lagging strand synthesis.

Answer 13

DNApolymeraseIIIbeginssynthesizingDNAinthe5’to 3’direction,beginningatthe3’endofeachRNAprimer.Thenewlysynthesizedstrandiscomplementaryandantiparalleltotheparentalstrandusedasatemplate.Thisstrandcanbemadecontinuouslyinonelongpieceandisknownasthe“leadingstrand”

2. The“laggingstrand”issynthesizeddiscontinuouslyasaseriesofsmallfragments(about1000nucleotideslong)knownasOkazakifragments.EachOkazakifragmentisinitiatedbythesynthesisofanRNAprimerbyprimase,andthencompletedbythesynthesisofDNAbyDNApolymeraseIII.Eachfragmentismadeinthe5’to 3’direction
3. dNTP todNMP and2Pidrivessynthesis 4.RNAprimersareremoved
   - accompanied by proofreading functions and Topoisomerases crucial for alleviating supercoiling

Question 14

What is the difference between the two types of Topoisomerases?

Answer 14

• 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

Question 15

What are some mechanisms for Topoisomerases II inhibition and what are they used for?

Answer 15

1.AlleviationofDNAsupercoilingbytopoisomerasesandareimportantdrugtargets.AlltopoisomeraseII-directedagentsareabletointerferewithatleastonestepofthecatalyticcycle.AgentsabletostabilizethecovalentDNAtopoisomeraseIIcomplex(alsoknownasthecleavablecomplex)aretraditionallycalledtopoisomeraseIIpoisons,whileagentsactingonanyoftheotherstepsinthecatalyticcyclearecalledcatalyticinhibitors.WhereastopoisomeraseIIpoisonsareusedsolelyfortheirantitumoractivities,catalytic
inhibitorsareutilizedforavarietyofreasons,includingtheiractivityas
antineoplasticagents(aclarubicin),cardioprotectors (ICRF-187),ormodulatorsinordertoincreasetheefficacyofotheragents(suramin andnovobiocin).

Question 16

How does DNA proofreading work?

Answer 16

When DNA polymerase mispairs a nucleotide with the template it uses 3’ > 5’ exonuclease activity to excise the mismatched nucleotide

Question 17

Describe DNA replication termination.

Answer 17

• 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

Question 18

What are three things that insure genome fidelity?

Answer 18

1. Geometry of active site of DNA polymerase
2. Proofreading - 3’ > 5’ exonuclease
3. Mismatch repair and DNA methylation

-estimated that less than one mutation introduced per cell division

Question 19

Describe the different prokaryotic DNA polymerases.

Answer 19

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)