3rd Week Course Notes - DNA Replication PDF

DEN230-5 Medical Biology DNA Replication Dr. Murat Kavruk [email protected] Room no:9911 1 Today’s Topics 1 Compare the roles of DNA polymerases in E. coli with those in mammalian cells. 2 Contrast the mechanisms of synthesis of the leading and lagging strands of DNA. 3 Identify the prot...

DEN230-5 Medical Biology DNA Replication Dr. Murat Kavruk [email protected] Room no:9911 1 Today’s Topics 1 Compare the roles of DNA polymerases in E. coli with those in mammalian cells. 2 Contrast the mechanisms of synthesis of the leading and lagging strands of DNA. 3 Identify the proteins found at replication forks of bacteria and mammalian cells. 4 5 6 Describe the mechanisms that ensure accurate DNA replication. Compare origins of replication in bacteria and mammalian cells. Summarize the action of telomerase 2 A) DNA Replication Follows a Set of Fundamental Rules DNA REPLICATION-1 A.1 DNA Replication is Semiconservative A.2 Regulation begins at Origin and Usually Proceeds Bidirectionally A.3 DNA Synthesis Proceeds in a 5’ to 3’ Direction and Semidiscontinuous B) DNA is Degraded by Nucleases C) DNA is synthesized by DNA Polymerases D) Replication is very Accurate E) E. coli Has at Least Five DNA Polymerases 3 F) DNA ReplicationDNA Requires Many Enzymes and Protein Factors REPLICATION-2 G) Replication of the E. coli Chromosome Proceeds in Stage Initiation Elongation Termination H) Bacterial Replication is Organized in Membrane-Bound Replication Factories I) Replication in Eukaryotic Cells is More Complex 4 All DNA polymerases.. 1) synthesize DNA only in the 5′ to 3′ direction, adding a dNTP to the 3′ hydroxyl group of a growing chain. 2) can add a new dNTP only to a preformed primer strand that is hydrogen-bonded to the template. 5 DNA Replication is Semiconservative Challenge: What is the pattern of replication? Observation: Semiconservative. Each initial strand become complementary with a completely new strand Regulation begins at Origin and Usually Proceeds Bidirectionally Following the confirmation of a semiconservative mechanism of replication, a host of questions arose: 1. Are the parent DNA strands completely unwound before each is replicated? 2. Does replication begin at random places or at a unique point? 3. After initiation at any point in the DNA, does replication proceed in one direction or both? 7 Challenge • Circular and linear genomes Observation • Replication Fork (radioactive thymidine) 8 Same for Eukaryotes An unlabeled chromosome proceeds through the cell cycle in the presence of 3Hthymidine 9 Regulation begins at Origin and Usually Proceeds Bidirectionally • DNA molecules in the process of being replicated contain Yshaped junctions called replication forks. • Two replication forks are formed at each replication origin. • At each fork, a replication machine moves along the DNA, opening up the two strands of the double helix and using each strand as a template to make a new daughter strand. • The two forks move away from the origin in opposite directions, unzipping the DNA double helix and copying the DNA as they go. 10 Regulation begins at Origin and Usually Proceeds Bidirectionally • DNA replication in both bacterial and eukaryotic chromosomes — is therefore termed bidirectional. • The forks move very rapidly: at about 1000 nucleotide pairs per second in bacteria and 100 nucleotide pairs per second in humans. • The slower rate of fork movement in humans (indeed, in all eukaryotes) may be due to the difficulties in replicating DNA through the more complex chromatin structure of eukaryotic chromosomes 11 Origin of Replication and Replicons Replicon: the length of DNA that is replicated following one initiation event at a single origin. Prokaryotes: Eukaryotes: • Single origin of replication • Multiple origion replications • Replicon represents whole genomes (~4.6 million bases for E. coli) • Multiple replicons represents whole genomes (~6.3 gigabases for humans) 12 5’ Initiation - Forming the Replication Eye oforReplication Bubble Origin 3’ 3’ 5’ Replication eye or replication bubble 3’ 5’ 5’ 3’ 3’ 5’ 5’ 5’ 3’ 3’ 5’ 3’ 3’ 5’ 5’ 3’ 13 DNA Polymerases First found in 1957: E. coli DNA polymerase I Need of all four types of dNTP and a DNA template 14 Bacterial DNA Polymerases DNA Pol III: responsible for the 5′ to 3′ polymerization essential for replication DNA Pol I: responsible for removing the primer, as well as for the synthesis that fills gaps produced after this removal. DNA Pol II, IV, V: DNA Pol II, as well as DNA Pol IV and V, are involved in various aspects of repair of DNA that has been damaged by external forces, such as ultraviolet light 15 A new strand of DNA is always synthesized in the 5’ to 3’ direction, with the free 3’ OH as the point at which the DNA is elongated. The movement of a replication fork is driven by the action of the replication machine, at the heart of which is an enzyme called DNA polymerase. This enzyme catalyzes the addition of nucleotides to the 3ʹ end of a growing DNA strand, using one of the original, parental DNA strands as a template. 16 Prokaryotes 1. Initiation 2. Helicase 3. RNA primer 4. Polymerization Eukaryotes 17 1. The helix must undergo localized unwinding, and the resulting “open” configuration must be Basic Steps of Replication stabilized so that synthesis may proceed along both strands. 2. As unwinding and subsequent DNA synthesis proceed, increased coiling creates tension further down the helix, which must be reduced. 3. A primer of some sort must be synthesized so that polymerization can commence under the direction of DNA polymerase III. Surprisingly, RNA, not DNA, serves as the primer. 4. Once the RNA primers have been synthesized, DNA polymerase III begins to synthesize the DNA complement of both strands of the parent molecule. 5. The RNA primers must be removed prior to completion of replication. The gaps that are temporarily created must be filled with DNA complementary to the template at each location. 6. The newly synthesized DNA strand that fills each temporary gap must be joined to the adjacent strand of DNA. 7. While DNA polymerases accurately insert complementary bases during replication, they are not perfect, and, occasionally, incorrect nucleotides are added to the growing strand. A proofreading mechanism that also corrects errors is an integral process during DNA synthesis. 1. Unwinding of DNA Helix • oriC region (9mers and 13mers of repetitive sequences) • DnaA protein binds 9mers region destabilize the helix • DNA helicase enzyme (in replication fork complex) binds to open helix and produce 2 ssDNA strands • ssDNA proteins binds to stabilize ssDNA’s 19 2. Decreasing the Tension • Unwinding creates twisting tension on DNA helix down the path (supercoiling) • Such supercoiling can be relaxed by DNA gyrase, a member of a larger group of enzymes referred to as DNA topoisomerases. • These various reactions are driven by the energy released during ATP hydrolysis 20 3. Initiation with RNA Primer • Short segment of RNA (about 10 to 12 nucleotides long), complementary to DNA, is first synthesized on the DNA template. • Synthesis of the RNA is directed by a form of RNA polymerase called primase, which is recruited to the replication fork by DNA helicase, and which does not require a free 3′ end to initiate synthesis. • Later, the RNA primer is clipped out and replaced with DNA. This is thought to occur under the direction of DNA Pol I. (step 5) 21 4. Continuous and Discontinuous DNA Synthesis • One strand is synthesized continuously and the other discontinuously. • The continuous strand; leading strand and the discontinuous strand; lagging strand which is in the direction opposite to the direction of fork movement. • Okazaki fragments range in length from a few hundred to a few thousand nucleotides, depending on the cell type. • Joining the fragments is the work of another enzyme, DNA ligase, which is capable of catalyzing the ligation of Okazaki fragments. (step 6) 22 23 5 and 6. Primer Removal and Ligation • In this process, an RNA or DNA strand paired to a DNA template is simultaneously degraded by the 5 ’ 3’ exonuclease activity of DNA polymerase I and replaced by the polymerase activity of the same enzyme. • These activities have a role in both DNA repair and the removal of RNA primers during replication. • DNA synthesis begins at a nick (a broken phosphodiester bond, leaving a free 3 hydroxyl and a free 5 phosphate). • Polymerase I extends the nontemplate DNA strand and moves the nick along the DNA, a process called nick translation. • A nick remains where DNA polymerase I dissociates, and is later sealed by DNA Ligase. 24 7. Proofreading Exonucleases degrade nucleic acids from one end of the molecule. Many operate one strand of a double stranded nucleic acid or of a single-stranded DNA. 5’ to 3’ Exonuclease activity 3’ to 5’ Exonuclease activity (Proofreading) Endonucleases can begin to degrade at specific internal sites in a nucleic acid strand or molecule, reducing it to smaller and smaller fragments • 1 in every 107 nucleotide pairs it copies is mispaired. • Proofreading takes place at the same time as DNA synthesis. • Before the enzyme adds the next nucleotide to a growing DNA strand, it checks whether the previously added nucleotide is correctly base-paired to the template strand. • If so, the polymerase adds the next nucleotide; if not, the polymerase clips off the mispaired nucleotide and tries again. • All DNA polymerases is a separate 3’ to 5’ exonuclease activity that double-checks each nucleotide after it is added. • The 3’ to 5’ exonuclease activity removes the mispaired nucleotide, and the polymerase begins again. This activity, known as proofreading. 26 27 8. (bonus) Termination • The two replication forks of the circular E. coli chromosome meet at a terminus region containing multiple copies of a 20 bp sequence called Ter (for terminus) (Fig. 25–17a). • The Ter sequences are arranged on the chromosome to create a sort of trap that a replication fork can enter but cannot leave. • The Ter sequences function as binding sites for a protein called Tus (terminus utilization substance). • The Tus-Ter complex can arrest a replication fork from only one direction. 28 Prokaryotes vs Eukaryotes Step in Replication Prokaryotic Cells Eukaryotic Cells Origin of replication One per chromosome Multiole per chromosome Unwinding of DNA double helix Helicase Helicase Stabiliation of ssDNA ssDNA-binding protein ssDNA-binding protein Synthesis of RNA primers Primase Primase Synthesis of DNA DNA Polymerase III DNA Polymerases α, δ, ε Removal of RNA primers DNA Polymerase I RNase Replacement of RNA with DNA DNA Polymerase I DNA Polymerase δ Joining of Okazaki fragments DNA Ligase DNA Ligase Removal of supercoils DNA gyrase DNA topoisomerases Synthesis of telomeres NA Telomerase Replication in Eukaryotic Cells Is More Complex • Origins of replication, called autonomously replicating sequences (ARS) or replicators, have been identified and best studied in yeast. • Yeast replicators span ~150 bp and contain several essential conserved sequences. About 400 replicators are distributed among the 16 chromosomes in a haploid yeast genome. • Initiation of replication in all eukaryotes requires a multisubunit protein, the origin recognition complex (ORC), which binds to several sequences within the replicator. 30 Termination Problem in Eukaryotes • Telomeric DNA is a simple repeat sequence with an overhanging 3′ end. Telomerase carries its own RNA molecule, which is complementary to telomeric DNA, as part of the enzyme complex. • The overhanging end of telomeric DNA binds to the telomerase RNA, which then serves as a template for extension of the template strand by one repeat unit. • The lagging strand of telomeric DNA can then be elongated by conventional RNA priming and DNA polymerase activity 31