Chpts 15 & 16DNA’s Primary Structure The primary structure of DNA has two major components:1. A backbone made up of the sugar and phosphate groups of deoxyribonucleotides2. A series of nitrogen-containing bases that project from the backboneDNA has directionalityOne end has an exposed hydroxyl group on the 3¢ carbon of deoxyriboseThe other end has an exposed phosphate group on a 5¢ carbonThe molecule thus has a 3¢ end and a 5¢ endDNA’s Secondary StructureWatson and Crick proposed two DNA strands line up in the opposite direction to each otherThis is called antiparallel fashion; the antiparallel strands twist to form a double helixThe secondary structure is stabilized by complementary base pairingDNA Strands Are Templates for DNA SynthesisWatson and Crick suggested the existing strands of DNA served as a template (pattern) for the production of new strandsBases were added to the new strands according to complementary base pairingThree alternative hypotheses for how the old and new DNA strands interacted during replication:1. Semiconservative replication2. Conservative replication3. Dispersive replicationHow Do the New DNA Strands Form? Three possibilities:In semiconservative replication, the parental DNA strands separate Each is used as a template for the synthesis of a new strandDaughter molecules each consist of one old and one new strand-In conservative replicationThe parental molecule serves as a template for the synthesis of an entirely new molecule-In dispersive replication:The parent molecule is cut into sections The daughter molecules contain old DNA interspersed with newly synthesized DNAA Comprehensive Model for DNA SynthesisMeselson and Stahl’s Key Experiment:Showed that each parental DNA strand is copied in its entirety; Semi-conservative replicationDid not illustrate a mechanism for this processDNA polymerase is the enzyme that catalyzes DNA synthesis The discovery of DNA polymerase cleared the way for understanding DNA replication reactionsCharacteristics of DNA Polymerases A critical characteristic of DNA polymerasesThey can work only in one directionDNA polymerases can add deoxyribonucleotides to only the 3′ end of a growing DNA chain DNA synthesis always proceeds in the 5′ ® 3′ directionDNA polymerization is exergonic because the monomers that act as substrates in the reaction are deoxyribonucleoside triphosphates (dNTPs)Have high potential energy because of their three closely packed phosphate groupsHow Does Replication Get Started?A replication bubble forms in a chromosome that is actively being replicated Grows as DNA replication proceedsBecause synthesis is bidirectional, in bacterial chromosomes, the replication process begins at a single location-- origin of replicationEukaryotes also have bidirectional replicationBut synthesis doesn’t start at the ends of chromosomes; rather, they have multiple origins of replication And they have multiple replication bubblesA replication fork is the Y-shaped region where the DNA is split into two separate strands for copyingHow Is the Helix Opened and Stabilized?Several proteins are responsible for opening and stabilizing the double helix:Enzyme helicase catalyzes the breaking of hydrogen bonds between the two DNA strands to separate themSingle-strand DNA-binding proteins (SSBPs) attach to the separated strands to prevent them from closingUnwinding the DNA helix creates tension farther down the helix, so the ezyme topoisomerase cuts and rejoins the DNA downstream of the replication fork, relieving this tension in the helixHow Is the Leading Strand Synthesized?DNA polymerase requires a free 3¢ hydroxyl (OH) group to commence, and there isn’t one availableSo DNA polymerase requires a primer to start this DNA replication processA few nucleotides possessing free 3¢ hydroxyl (OH) groups are bonded to the template strand; this is the primer…it is made of RNA because RNA polymerase doesn’t require a primer…This provides a free 3¢ hydroxyl (OH) group that can combine with an incoming dNTP to form a phosphodiester bond; later the RNA segmentis removedPrimaseA type of RNA polymeraseSynthesizes a short RNA segment that serves as a primerDNA polymerase III then adds bases to the 3¢ end of the primerThe product is called the leading strand, or continuous strandIt leads into the replication fork The Lagging StrandThe other DNA strand is called the lagging strand It is synthesized discontinuously in the direction away from the replication forkIt occurs because DNA synthesis must proceed in the 5¢ ® 3¢ directionHow Is the Lagging Strand Synthesized?Synthesis of the lagging strand starts when Primase synthesizes a short stretch of RNA again acting as a primer DNA polymerase III then adds bases to the 3¢ end of the primerDNA polymerase moves away from the replication fork Helicase continues to open the replication fork And expose single-stranded DNA on the lagging strandThe Discontinuous Replication HypothesisOnce primase synthesizes an RNA primer on the lagging strand,DNA polymerase might synthesize short fragments of DNA along the lagging strandThese fragments would later be linked together to form a continuous whole strandThis hypothesis was tested by Okazaki and his colleaguesThe Discovery of Okazaki FragmentsThe lagging strand is synthesized as short discontinuous fragments called Okazaki fragmentsDNA polymerase I then removes the RNA primer at the beginning of each Okazaki fragment Fills in the gap The enzyme DNA ligase joins the Okazaki fragments to form a continuous DNA strandBecause Okazaki fragments are synthesized independently and joined together later, the lagging strand is also called the ‘discontinuous strand’DNA Synthesis Enzymes Are Well-OrganizedThe replisome: Contains enzymes responsible for DNA synthesis around the replication fork Joined into one large, multi-enzyme machineReplicating the Ends of Linear ChromosomesTelomere is the region at the end of a linear chromosome Does not contain genesConsists of short, repeating stretches of basesReplication of telomeres can be problematicLeading-strand synthesis results in a normal copy of the DNA molecule, but the telomeres (regions at the ends of linear chromosomes) on thelagging strand shortens during DNA replicationReplication fork reaches the end of a linear chromosome Because there is no available primer for DNA synthesis, there is no way to replace the RNA primer from the lagging strand with DNA The primer is removed,