Chpts 15 16 DNA s Primary Structure The primary structure of DNA has two major components 1 A backbone made up of the sugar and phosphate groups of deoxyribonucleotides 2 A series of nitrogen containing bases that project from the backbone DNA has directionality One end has an exposed hydroxyl group on the 3 carbon of deoxyribose The other end has an exposed phosphate group on a 5 carbon The molecule thus has a 3 end and a 5 end DNA s Secondary Structure Watson and Crick proposed two DNA strands line up in the opposite direction to each other This is called antiparallel fashion the antiparallel strands twist to form a double helix The secondary structure is stabilized by complementary base pairing DNA Strands Are Templates for DNA Synthesis Watson and Crick suggested the existing strands of DNA served as a template pattern for the production of new strands Bases were added to the new strands according to complementary base pairing Three alternative hypotheses for how the old and new DNA strands interacted during replication 1 Semiconservative replication 2 Conservative replication 3 Dispersive replication How 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 strand Daughter molecules each consist of one old and one new strand In conservative replication The 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 DNA A Comprehensive Model for DNA Synthesis Meselson and Stahl s Key Experiment Showed that each parental DNA strand is copied in its entirety Semi conservative replication Did not illustrate a mechanism for this process DNA polymerase is the enzyme that catalyzes DNA synthesis The discovery of DNA polymerase cleared the way for understanding DNA replication reactions Characteristics of DNA Polymerases A critical characteristic of DNA polymerases They can work only in one direction DNA polymerases can add deoxyribonucleotides to only the 3 end of a growing DNA chain DNA synthesis always proceeds in the 5 3 direction DNA 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 groups How Does Replication Get Started A replication bubble forms in a chromosome that is actively being replicated Grows as DNA replication proceeds Because synthesis is bidirectional in bacterial chromosomes the replication process begins at a single location origin of replication Eukaryotes also have bidirectional replication But synthesis doesn t start at the ends of chromosomes rather they have multiple origins of replication And they have multiple replication bubbles A replication fork is the Y shaped region where the DNA is split into two separate strands for copying How 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 them Single strand DNA binding proteins SSBPs attach to the separated strands to prevent them from closing Unwinding 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 helix How Is the Leading Strand Synthesized DNA polymerase requires a free 3 hydroxyl OH group to commence and there isn t one available So DNA polymerase requires a primer to start this DNA replication process A 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 segment is removed Primase A type of RNA polymerase Synthesizes a short RNA segment that serves as a primer DNA polymerase III then adds bases to the 3 end of the primer The product is called the leading strand or continuous strand It leads into the replication fork The Lagging Strand The other DNA strand is called the lagging strand It is synthesized discontinuously in the direction away from the replication fork It occurs because DNA synthesis must proceed in the 5 3 direction How 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 primer DNA polymerase moves away from the replication fork Helicase continues to open the replication fork And expose single stranded DNA on the lagging strand The Discontinuous Replication Hypothesis Once primase synthesizes an RNA primer on the lagging strand DNA polymerase might synthesize short fragments of DNA along the lagging strand These fragments would later be linked together to form a continuous whole strand This hypothesis was tested by Okazaki and his colleagues The Discovery of Okazaki Fragments The lagging strand is synthesized as short discontinuous fragments called Okazaki fragments DNA 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 strand Because Okazaki fragments are synthesized independently and joined together later the lagging strand is also called the discontinuous strand DNA Synthesis Enzymes Are Well Organized The replisome Contains enzymes responsible for DNA synthesis around the replication fork Joined into one large multi enzyme machine Replicating the Ends of Linear Chromosomes Telomere is the region at the end of a linear chromosome Does not contain genes Consists of short repeating stretches of bases Replication of telomeres can be problematic Leading strand synthesis results in a normal copy of the DNA molecule but the telomeres regions at the ends of linear chromosomes on the lagging strand shortens during DNA replication Replication 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 leaving a section of single stranded DNA lagging