Presentation on theme: "DNA Replication 1-General Principles"— Presentation transcript:
1 DNA Replication 1-General Principles 2-The enzymology of DNA polymerasesA-General Properties:B-Bidirectionality and Priming problemsC-Catalytic Properties of DNA polymerase IPolymerase5’->3’ exonuclease3’->5’ exonuclease and proofreading3- Prokaryotic DNA ReplicationA - The prokaryotic ReplisomeB - Interference between Replication and Transcription• Not treated:Old experiments (Meselson & Stahl,Cairns)4- Eukaryotic DNA ReplicationA-The eukaryotic ReplisomeB- Dealing with ChromatinC-Dealing with linear chromosomes:Telomeres and Telomerase
2 DNA Replication by DNA polymerases : Copying the genetic material to prepare for cell division ParentalDNAReplication is Semi-Conservative:1 parental strand is transmitted into eachdaughter DNA molecule (Meselson & Stahl exp.)2 CopiesReplication is bidirectional from theOrigin of Replication:(Cairns exp.)• 1 origin of replication in most eubacterial chromosomes• several origins of replications inarchea, some eubacteria, and in eukaryotes
3 Fundamental properties of DNA Polymerases BasePOHO-Base n5’HOBase n+1:+5’3’primertemplatedNTPs5’3’New strandPPiCatalyze the polymerization of deoxyribonucleotidesin the 5’->3’ direction: (dNMP)n+dNTP -> (dNMP)n+1 + PPi2) Require a template (usually DNA)3) Require a primer (DNA or RNA)
4 The bidirectionality problem Synthesis of DNA on thelagging strand requirescontinuous synthesisof primersSynthesis of DNAis semi-discontinuous
5 Where do the primers used during DNA replication come from ? The primers are made of RNA since RNA polymerasesdo not require primers. The existence of joint RNA-DNAmolecules was demonstrated by alkaline hydrolysisof Okazaki fragments.DNAsynthesisBBOHPBOHPPPPPPOHOHrNTPsa32P-dNTPsAlkalinehydrolysisBBBBBBBOHOHOHOHOHOHOHPPPPPPRibonucleotide with a 3’P-: Diagnostic of a 5’RNA-3’DNAjunctionalkaline hydrolysis
6 DNA Polymerase I = the prototype DNA polymerase - Discovered in the late ‘50s by Arthur Kornberg(1959 Nobel Prize in Medicine)- First DNA polymerase discovered- 3 Enzymatic activities associated to three distinct active sites on a single polypeptide chainThe activities can be artificially separated by experimental treatment with the trypsin protease( these fragments have no physiological relevance)109 kD+NH3COO-Limited Tryptic proteolysis34 kD75 kD = Klenow Fragment5’ -> 3’Exonuclease5’ -> 3’ Polymerase3’ -> 5’ Exonuclease
7 (T.Steitz) Two metal ion mechanism for DNA Polymerase I - Me=divalent metal ion(usually Mg++)MeA=activates the 3’OH forattack on the a phosphate of theincoming dNTP (lowers pKa of 3’O)MeB=plays the dual role of stabili-zing the neg.charge that builds upon the leaving oxygen and chelatingthe and phosphatesMeA and B stabilize both the structureand charge of the pentacovalenttransition state
8 How does DNA Polymerase I select correct nucleotides from incorrect nucleotides ? formingnon Watson-Crickbase pairs do not fitthe active site andare ejectedSuggests thatH-bonding per sedoes not contributeto nt selection byDNA polymerasesNucleotides formingWatson-Crickbase pairs fitthe active site(BLUE SQUARE)
9 select the proper nucleotide: Testing the importance How do DNA polymerasesselect the proper nucleotide:Testing the importanceof H-bondingin base pairsfor the fidelity ofnucleotideincorporationThymineDi-fluorotolueneCan H-bond with ASame size asThymine butcannot H-bond with A5’3’XTemplate = 24 ntLabeled primer= 23 ntAfter extension = 24 ntA,C,G,Tor F ?
10 Discrimination for deoxynucleotides vs ribonucleotides by DNA polymerasesAlignment of sequences of polymerases active siteUse dNTPUse riboNTPUse dNTPUse riboNTPCatalytic AspThe aromatic/large side chain found in DNA polymerasesclose to the dNTP binding site provides a steric gate against riboNTPsSteric Clashwith the Y416residue if a 2’-OHis presentBiochemistry, 41, 10256–10261
11 3’-> 5’ exonuclease activity of DNA Polymerase I OHO-Base n(now last base)5’Base n+1(Last Base of DNA)H:-O3’-> 5’exonucleaseactivityof DNAPolymerase IThis reaction is not the reversal of the 5’->3’ polymerization:The attacking group is water rather than pyrophosphate(a hydrolysis rather than a pyrophosphorolysis).For this reason, the active sites of the polymerization and of the3’->5’ exonuclease reactions must be different. This is essential forthe biological role of the 3’->5’ exonucleolytic reaction, which is toedit newly polymerized sequences.
12 e P2 = P1 Not making mistakes during polymerization is thermodynamically impossible5’3’A5’templateP1P25’T5’C3’A5’3’A5’templatetemplateP2 = probability of incorporatingone incorrect nucleotidee.g. A C mismatchP1 = probability of incorporatingthe right nucleotideA:T base pairP2P1=e(DGAxC DGA:T) / RT(derived from the Boltzman distribution)P2DGAxC DGA:T= 3 kcal/mol.=0.01 or 1% - at leastP1DNA polymerases are much more accurate than HOW ??
13 Editing of newly synthesized DNA by the 3’->5’ exonuclease activity templatePolymerization5’3’5’template3’->5’ exo triggeredby the mistakeNewstrand5’3’5’3’->5’ exo5’3’5’Polymerization3’5’5’Editing of mistakes require a switch between :polymerization mode and editing mode
14 Switch between Polymerizing and Editing Modes in DNA Polymerase: Structural Basis for “Proofreading”
15 5’-> 3’ exonuclease activity of DNA Polymerase I OHO-Base 1Base 25’end-OH:(now base 1)New 5’endBiological significance:Allows the replacement ofdamaged or abnormal DNAsequences by “Nick translation”(important for DNA RepairChapter)Also allows the removal of RNAsequences embedded in DNA(removal of replication primers).dNTPs PPiThe nick has moved“bad”DNAnickNew strand5’3’5’3’3’5’3’5’
16 The replisome of E.coli 1) Helicases 6) DNA topoisomerase II Unwind DNA at the replication forkin a reaction coupled to ATP Hydrolyis2) Single-stranded DNAbinding proteins (SSB)Bind and stabilize the DNA in a singlestranded conformation after the meltingby helicases3) The PrimosomeSynthesizes RNA primersof the lagging strandContains Primase4) DNA Polymerase III :The replicase6) DNA topoisomerase IIRelaxes supercoiled DNA thatforms ahead of the replication fork.Decatenates the final product7) Rnase HRemoves RNA primers8) DNA Polymerase IReplaces RNA primers withDNA by nick translation8) DNA LigaseJoins the Okazaki fragments
17 The Replisome of E.coli in action “old model” Trends inMicrobiology15, (2007)DNA PolymeraseIII Core- 130 kD =Catalytic sitefor polymerizationkD =3’->5’ editingexonucleaseq kD = structural role ?b Subunit =Sliding ClampA homodimer of 2 X 41 kDATP-dependent processivityfactor = b clampPol.III Core is poorlyprocessive by itselfComplex= Clamp loadert SubunitThe Replisome ofE.coli in action“old model”4 polypeptidesATP-dependent conformational changesFacilitates the loading of the b clamponto DNADimerization factorHolds two Pol. IIIcores together
18 Sliding b clamps provide processivity to DNA polymerase III 3’b- ClampsNewlyReplicatedStrand5’homodimer of 2 X 41 kDwrapped around dsDNATemplate Strand3’
19 The replisome of E.coli in action: Cycles of molecular events during Lagging Strand synthesis (1) Step1: The primase synthesizesa new RNA primer upstream in thelagging strand; the two polymerasereplicate DNAStep2: A sliding clamp is assembledaround the new RNA primer;primase dissociatedTrends in Microbiology 15, (2007)
20 The replisome of E.coli in action: Cycles of molecular events during Lagging Strand synthesis (2) Step2: A sliding clamp is assembledaround the new RNA primer;primase dissociatedStep3: the lagging strand polymerasedetaches and associates with the newlydeposited sliding clampTrends in Microbiology 15, (2007)
21 The replisome of E.coli in action: Cycles of molecular events during Lagging Strand synthesis (3) Step3: the lagging strand polymerasedetaches and associates with the newlydeposited sliding clampStep4: the lagging strand polymerasestart to synthesize the next Okazakifragment; primase will reinitiatesynthesis of the next RNA primer back to Step1Trends in Microbiology 15, (2007)
22 The Clamp loader use cycles of ATP hydrolysis to open and load the sliding clamps around the primed DNABinding of theClamp loaderto b-clamps;Opening ofthe b-clamps;Loading theb-clamps ona primer-templateduplexRecyclingof the clamploaderKelch et al. - Science 23 December 2011: Vol. 334 no pp
23 Newsflash – Recent works shows that There are 3 core DNA polymerases associated with most replisomes in vivoNature Structural & Molecular BiologyVolume: 19, Pages: 113–116 (2012)What is the advantage of3 Polymerase replisomes vs 2 Polymerases ?Science. 2010328(5977): 498–501
24 Why is a triPolymerase replisome ? TriPol.IIIdiPol.III• TriPol.III replisome is more processive• TriPol.III replisome leaves less gaps tobe filled by Pol.I-> DNA Replication is overall more efficientNature Structural & Molecular BiologyVolume: 19, Pages: 113–116 (2012)
25 Problem of Coordinated Nucleic Acids Synthesis in vivo: What happens when DNA Polymerases and RNA polymerases collide ? (speed of DNA polymerase >> speed of RNA polymerase )Nature456,(2008)
26 Collision between DNA Polymerases and RNA polymerases result in polymerases dissociation and in the use of the RNA synthesized by RNAPolymerase as a primer for replicationNature 456, (2008)
27 Eukaryotic DNA Replication: it’s similar to bacterial DNA replication…. but it’s different !• Machinery is overall similar to that use for bacterial DNA replication (names are different…)• Idiosyncraties of eukaryotic DNA replication are linked to the size andorganization of eukaryotic genomes:• large size of eukaryotic chromosomes and limited time for DNAsynthesis requires multiple origins of replication• Replication machinery needs to deal withnucleosome packaging of eukaryotic DNA• Problem of linear chromosomes
28 Architecture of the Eukaryotic Replisome is similar to that of the bacterial ReplisomePol e - Replicates leading strandPol d - Replicates lagging strandPCNA:(proliferating cellsNuclear antigen):=trimeric sliding clampMCM =heterohexameric helicaseFEN1 = nuclease thatremoves RNA primersPol -primaseComplex containing both primaseand DNA polymerase a activitiesMol.Cell30,(2008)Replication Protein A (RPA) = SSB
29 The eukaryotic cell cycle Eukaryotic DNA Replication:The limited time for DNA replication (6-8 hours) combined to the increased size of the genomes (>107 base pairs) explain the requirement for multiple replication origins
30 Histone chaperones Eukaryotic Replication needs to deal with the nucleosome packaging of eukaryotic DNA• Needs to Remove Histones upstream from the replication fork such thatreplication is not impeded• Needs to reassemble histones/nucleosomes on newly replicated DNA tomaintain chromatin structure and epigenetic marksHistone chaperonesExample of Asf1, an H3/H4 histone chaperone that interacts with MCMMCMReplicationAsf1 remove H3/H4 histonesupstream from MCM and reloadsthem after replication has proceededH3/H4Asf1 can also add “new” H3/H4 histones since there is only ½ of the histones needed on the parental DNAScience 21 December 2007:Vol no. 5858, pp
31 Eukaryotic DNA Replication: Problem of maintaining the ends of linear chromosomes is linked to the degradation of RNA primerslast primers on each 5’-end are removed but the gaps cannot be filled because of the lack of 3’-OH groupEach cycle of replication would result inprogressive chromosome shortening
32 Preservation of Telomeres by the telomerase Telomerase (TERT):1 RNA subunit (template)several proteins:1 “reverse transcriptase”After extension of the upper strand by telomerase,the replication machinery can now use this strandto make a new RNA primer using primase, then a newokazaki fragment and fill in the lower strand to“elongate” this strand.
34 Connections between telomerase and aging HumansSomatic tissues lack telomerase, possibly contributing to the normal physiological symptoms of aging. Shortening of telomeres may lead to senescence in cultured human cellsStem cells, and cancer cells all contain telomerase activity possibly explaining their ability to divide indefinitely. Germ cells and early embryos also contain telomerase.MiceTelomerase protein or RNA mutant mice are fine for a few generations, perhaps because of the extraordinarily long telomeres in laboratory miceAfter a few generations, the telomerase-mutant mice exhibit reduced fertility, signs of premature aging, and shortened life-spanRudolph et al (1999) Longevity, stress response, and cancer in aging telomerase-deficient mice.Cell 96,The picture is of a 3rd generation telomerase RNA mutant mouse.Gonzalez-Suarez E, Geserick C, Flores JM, Blasco MA. (2005) Antagonistic effects of telomerase on cancer and aging in K5-mTert transgenic mice.Oncogene Jan 31;Prematurely grey, balding
35 Accelerated telomere shortening in response to life stress PNAS 101 (49), (2004)“Women with the highest levels ofperceived stress have telomeresshorter on average by the equivalentof at least one decade of additionalaging comparedto low stress women”.