The Chemistry of DNA synthesisDNA synthesis requires Deoxynucleoside triphosphates and a primer:template junctionRequirements1. dGTP, dCTP, dATP, dTTP2. Primer:template junction1. Provides the ssDNA that directs the addition of each complimentary deoxynucleotide (complimentary but shorter than the template)2. The primer must have an exposed 3’OH that will be extended as the new nucleotides are addedDNA is synthesized by extending the 3’ end of the primerFeature of both DNA and RNAPhosphodiester bond formed and the leaving group is pyrophosphateTemplate strand directs which nucleoside triphosphate is added (complimentary)Hydrolysis of pyrophosphate is the driving force for DNA synthesisAdditional free energy added by rapid hydroysis of the pyrophosphate into two phosphate groups by pyrophosphataseBreaks two high energy bondsThe Mechanism of DNA polymeraseDNA polymerases use a single active site to catalyze DNA synthesisDNA polymeraseUse single active site to catalyze the addition of any four deoxynucleoside triphosphatesKinetic selectivityDNA polymerases resemble a hand that grips the primer: template junctionLarge cleft like hand (thumbs, fingers, and palm)Palm domainB sheet and primary elements of catalytic siteMetal ionsMonitors base pairing of the most recently added nucleotidesFingersImportant for catalysisBind to incoming dNTP, moves to enclose dNTPThumbInteracts with DNA that has been most recently synthesized to maintain correct position of the primer and active site and to help maintain a strong association between the DNA polymerase to add many dNTPs each time it binds a primer:template junctionDNA polymerases are processive enzymesProcessiviyAverage number of nucleotides added each time the enzyme binds a primer:template junctionComtirbutes to why polymerase is rapidMultiple nucleotides per binding eventIncreases DNA synthesis by 1000 foldSequence independent nature of these interactions permits the easy movement of DNA after it binds to the polymeraseExonuclease proofread newly synthesized DNA1 in 10^5 times flickering of bases into the wrong tantomerc form-incorrect base pairs positionedproofreading corrects these mistakesproofreading exonucleasecapable of degrading DNA starting from 3’ DNA endpreference for degrading DNA containing incorrect base pairsThe Replication ForkBoth strands of DNA are synthesized together at the replication forkReplication forkHe junction between the newly separated template strands and the unreplicated duplex DNAMoves continuously towards the duplex region of unreplicated DNA leaving two ssDNA templateslagging strand/leading strandpg. 209synthesis of the lagging strand must wait for movement of the replication fork to expose a substantial length of template before it can be replicatedokazaki fragmentsthe resulting short fragments of new DNA formed on the lagging strand (1000-2000 nucleotides)transient intermediatesThe initiation of New strand od DNA requires a RNA primerAll DNA polymerases require a primer with a free 3’OHPrimaseSpecialized RNA polymerase dedicated to making short RNA primers on a ssDNA template which are extended by DNA polymeraseLeading strand requires one primerLagging strand needs new primer for each fragmentPrimase activity increased when it associates with DNA helicaseRNA primers must be removed to complete DNA replicationRNaseHRecognizes and removes most of each RNA primerSpecifically degrades RNA that is base paired with DNARemoves all RNA primer except for the ribonucleotide directly linked to DNA endFinal nucleotide is removed by and exonuclease that degrades RNA or DNA from their 5’ endRemoval of RNA primer creates gap that fits DNA polymerase very wellDNA ligaseRemoves nicks in DNAUses ATP to create phosphodiester bond between adjacent 5’ phosphate and 3’ OHDNA helicases unwind the double helix in advance of the replication forkDNA helicaseCatalyze the separation of the two strands of duplex DNAUse energy of ATPHexameric proteins encircle DNA]actAct processivelyPolarityEach helicase moves in a defined direction along the DNACan have either 3’-5’ polarity or 5’-3’DNA Hleicase pulls single stranded DNA through a central protein poreSix hands pulling on a rope in a hand over hand mannerSingle Stranded DNA binding proteins stabilize ssDNA prior to replicationSSBsStabilize separated strands by binding to themCooperative bindingSSB molecules bound to immediately adjacent regions of ssDNA also bind ot each otherDNA held in an elongated stateTopoisomerases remove supercoils produced by DNA unwinding at the replication forkPositive supercoiling in from of the forkTopoisomerases act as a swivelase that fix these problemsReplication fork enzymes extend the range of DNA polymerase substratesDNA helicase and isomerase perform functions without having to form new moleculesProteins act in a sequence independent mannerThe specialization of DNA polymerasesDNA polymerases are specialized for different roles in the cellDNA pol 3 holoenzymePart of a larger complex that confers very high processivityDNA polymerase 1Specialized for the removal of the RNA primers that are used to initiate DNA synthesis5’ exonuclease removal upstream of DNA synthesisnot highly processiveideal for RNA primer removalEukaryotic enzymesDNA pol sigmaHighly processiveLagging strandDNA pol epsilonHighly processiveLeading strandDNA pol alpha primaseInitiating new DNA strands4 subunitsrapidly replaced by other enzymesPolymerase switchingThe process of replacing DNA pol alpha primase with pol episolon or sigmaSliding clamps dramatically increase DNA polymerase processivityProcessivity increased at replication forkSliding DNA clampsShape of doughnutSlide along DNAMake sure the DNA polymerase rapidly rebinds the same primer:template jnction increasing the processivity of the DNA polymeraseSliding clamps are opened and placed on DNA by clamp loadersSliding clamp holdersCatalyze the opening and placement of sliding clamps on the DNAATP binding and hydrolysisAlso removes clamps when they are no longer in useAlter conformation of target but not chemical conformationDNA synthesis at the replication forkLeading and lagging strands synthesized simultaneouslyThis limits the amount of ssDNA present in the cell during DNA replicationDon’t want a lot of ssDNA present for a long period of timeMultiple DNA polymerases function at the replication forkHoloenzymeGeneral name for a miltiprotein complex in which a core enzyme activity is associated with