HSS2305A Spring 2022 DNA Replication Lecture Notes PDF

HSS2305: Molecular Mechanisms of Disease Lecture 13 – DNA Replication, DNA Damage and DNA Repair Prof. Keir Menzies  DNA Replication Reproduction is a property of all organisms Genetic material must be passed on DNA must therefore be copied during mitosis and meio...

HSS2305: Molecular Mechanisms of Disease Lecture 13 – DNA Replication, DNA Damage and DNA Repair Prof. Keir Menzies  DNA Replication Reproduction is a property of all organisms Genetic material must be passed on DNA must therefore be copied during mitosis and meiosis DNA replication Copying of DNA Replication machinery is also used for DNA repair Human diploid cells have 46 chromosomes (gametes/haploid have 23)  DNA Replication Semi-Conservative Replication: Watson & Crick Gradual separation of the strands of the double helix Hydrogen bonds between strands broken Zipper Synthesis of two daughter strands complementary to the two parental templates Each daughter duplex contains one strand from the parent structure DNA Replication Bacterial Heavily studied Temperature sensitive mutants (allows us to turn gene expression on and off quickly -enabling the study of essential genes and proteins that would be lethal if permanently inactivated) In vitro culture systems > 30 proteins involved in DNA replication Prokaryotes (bacteria) and eukaryotic cells have similar DNA replication DNA Replication https://www.youtube.com/watch?v=TNKWgcFPHqw  DNA Replication Eukaryotes S-Phase ”Synthesis” Phase Cell cycle phase where DNA is replicated Replicons Many small portions of eukaryotic genome are replicated (replicons) all at once Origin Replicon’s initiation site Human cells 10,000-100,000 different origins ~ 10-15% replicons actively engaged during S phase  DNA Replication Eukaryotes S-Phase ”Synthesis” Phase Cell cycle phase where DNA is replicated Replicons Many small portions of eukaryotic genome are replicated (replicons) all at once Origin Replicon’s initiation site Human cells 10,000-100,000 different origins ~ 10-15% replicons actively engaged during S phase  DNA Replication Eukaryotes Replication proceeds both directions “bi-directionally” Replication fork Site where: Parental double helix is undergoing strand separation Nucleotides incorporated into new complementary strand 2 replication forks move in opposite directions  DNA Replication Eukaryotes: Nuclear Structure Replication foci localized sites within the nucleus in which several replicons have become active ~ 50-250 replication foci/replicating nucleus ~10-100 active replication forks (replicons)/replication foci  DNA Replication Prokaryotes: DNA Polymerase DNA polymerase holoenzyme: Enzymes responsible for new DNA synthesis A DNA Polymerase III dimer synthesizes the daughter (leading and lagging) strands simultaneously with other proteins (called DNA Polymerase Holoenzyme) Slightly misleading since it Daughter strands = newly formed strands appears to show the 3 requirements of DNA Polymerase dimer of DNA polymerase 1. Template DNA strand to copy working separately: see 2. RNA Primer strand to which nucleotides can be added next slides DNA Polymerase I replaces RNA primer with DNA 3. Only synthesizes new DNA in a 5`→ 3` direction (direction of new daughter strand) 3’OH group carries out a nucleophilic attack on 5`α- phosphate of incoming nucleoside triphosphate (DNA polymerase III holoenzyme and DNA polymerase I are the most important polymerases for replication in prokaryotes) https://www.youtube.com/watch?v=9oxwN71oACI  DNA Replication Semi-Discontinuous Semi-discontinuous replication: New DNA daughter strand created in 5’ -> 3’ direction Leading strand Grows continuously towards the replication fork Lagging Strand Second strand grows discontinuously away from fork Okazaki fragments Small DNA fragments Each fragment requires a small RNA primer (in green) to begin Reduces mismatch error  DNA Replication Prokaryotes: Initiation Bacterial Cells DnaA recognizes orgin of replication (not seen in diagram) DnaB helicase: Unwinding enzyme Binds to origin (OriC) which in bacteria is just DnaB one site Uses ATP hydrolysis to break hydrogen bonds between strands Separates DNA strands Single-stranded DNA-binding (SSB) proteins Coat the unwound DNA Primase Synthesizes RNA Primers Makes foundation for Okazaki fragments RNA primers extended by DNA polymerase III (as part of the DNA polymerase holoenzyme)  DNA Replication Eukaryotes: Initiation Eukaryotic cells Origin recognition complex (ORC) recognizes the origin of a replicon 6 proteins make this complex Licensing factors Licensing factors Cdc6 and Cdt1 Recruited to origin Recruit helicase Helicase Helicase Unwinding protein Comprised of minichromosome maintenance proteins (MCM2-7)  DNA Replication Eukaryotes: Initiation Eukaryotic cells Pre-replication Complex (pre-RC) ORC complex, licensing factors & helicase bind during G1 Protein kinases (Cdk, DDK) phosphorylate and activate pre- RC High activity in S phase P Cdk They also inhibit formation of new Pre-RCs helps to synchronize replication New Pre-RCs form only in M and G1 Primase (in preparation for new round) Primase Synthesizes RNA Primers Makes foundation for Okazaki fragments  DNA Replication Supercoiling DNA Supercoiling Occurs during DNA unwinding Built up DNA tension http://youtu.be/k4fbPUGKurI In eukaryotes: Type I topoisomerases Relaxes DNA (i.e., remove coils) by nicking and closing one strand of duplex DNA. It allows rotation around the intact strand to relieve strain during transcription or replication. Does not require ATP Type II topoisomerases Change DNA topology by breaking and rejoining double-stranded DNA Introduce or remove supercoils This action is critical for untangling and separating intertwined DNA strands, especially during replication and cell division​ Requires ATP In Bacteria: DNA Gyrase  DNA Replication Prokaryotes: Elongation DNA Polymerase III holoenzyme: 2 core DNA III polymerases → replicate DNA ≥2 β clamps → allow polymerase to remain associated with DNA 1 clamp on leading strand 1 clamp/Okazaki fragment on lagging strand * γ-Clamp loading complex → loads sliding clamp onto DNA, also bound to helicase * Replisome * Refers to the entire complex of active proteins at replication fork DNA pol III holoenzyme, helicase, SSBs, primase DNA Replication Elongation  DNA Replication Prokaryotes : Elongation Helicase → unwinds and located on lagging strand Primase → synthesizes RNA primers on lagging strand 1. DNA polymerase III → extends RNA primers on lagging strand Attaches to RNA primer and incorporates deoxynucleotides Lagging strand looped so that 2 DNA polymerases can travel together (Note: DNA Polymerase II is not as well described but has been described as a backup polymerase for DNA polymerase III)  DNA Replication Prokaryotes : Elongation Helicase → unwinds and located on lagging strand Primase → synthesizes RNA primers on lagging strand 1. DNA polymerase III → extends RNA primers on lagging strand Attaches to RNA primer and incorporates deoxynucleotides) Lagging strand looped so that 2 DNA polymerases can travel together (Note: DNA Polymerase II is not as well described but has been described as a backup polymerase for DNA polymerase III)  DNA Replication Prokaryotes : Elongation Helicase → unwinds and located on lagging strand Primase → synthesizes RNA primers on lagging strand 1. DNA polymerase III → extends RNA primers on lagging strand Attaches to RNA primer and incorporates deoxynucleotidesLagging strand looped so that 2 DNA polymerases can travel together (Note: DNA Polymerase II is not as well described but has been described as a backup polymerase for DNA polymerase III)  DNA Replication Prokaryotes : Elongation Helicase → unwinds and located on lagging strand Primase → synthesizes RNA primers on lagging strand 1. DNA polymerase III → extends RNA primers on lagging strand Attaches to RNA primer and incorporates deoxynucleotides Lagging strand looped so that 2 DNA polymerases can travel together (Note: DNA Polymerase II is not as well described but has been described as a backup polymerase for DNA polymerase III)  DNA Replication Prokaryotes : Elongation 2. DNA Polymerase III releases lagging-strand once it encounters previously synthesized Okazaki fragment 3. DNA Polymerase III binds the lagging strand template further along its length and elongates DNA from next RNA primer Trombone Model https://youtu.be/4jtmOZaIvS0  DNA Replication Prokaryotes : Elongation 2. DNA Polymerase III releases lagging-strand once it encounters previously synthesized Okazaki fragment 3. DNA Polymerase III binds the lagging strand template further along its length and elongates DNA from next RNA primer Trombone Model https://youtu.be/4jtmOZaIvS0  DNA Replication Prokaryotes : Elongation 4. DNA polymerase I Exonuclease activity (5`→3`) removes RNA primers of Okazaki fragment, polymerase activity fills gap with dNTs 5. DNA ligase Covalently joins 3’ dNT to 5` end of Okazaki fragment  DNA Replication Elongation Eukaryotic Replication Fork:  DNA Replication Elongation Eukaryotic Replication Fork:  DNA Replication Elongation Eukaryotic Replication Fork:  DNA Replication Elongation Eukaryotic Replication Fork:  DNA Replication Elongation Eukaryotic Replication Fork: (clamp)  DNA Replication Elongation Eukaryotic Replication Fork:  DNA Replication Eukarotes S-Phase ”Synthesis” Phase Cell cycle phase where DNA is replicated Replicons Small portions of eukaryotic genome that are replicated Origin Replicon’s initiation site Human cells 10,000-100,000 different origins ~ 10-15% replicons actively engaged during S phase  DNA Replication Eukaryotes: Chromatin Structure Movement of replication machinery along the DNA displaces nucleosomes Reassembly of nucleosomes on daughter strand very quick Histone molecules used for this process are from parental chromosomes and newly synthesized (H3H4)2 tetramers → remain intact H2A/H2B dimers → separate and bind randomly to new and old tetramers  DNA Replication and Human Disease > 160 different proteins involved in replicating the human genome ~ 80 genetic diseases mutations in these proteins errors in DNA replication or repair Example below shows diseases in mutations of different helicase proteins: Don’t need to memorize!  DNA Repair high fidelity Spontaneous mutation rate error rate of incorporating an incorrect nucleotide during DNA replication Incorporation of a particular nucleotide depends upon geometry only one dNT forms a proper geometric fit with template This dNT fits into a binding pocket within DNA polymerase Angle is not correct if : adenine (A) with thymine (T), and cytosine (C) with guanine (G)  DNA Repair Prokaryotes: Mismatch repair DNA Polymerases I and III have 3’->5’ exonuclease proofreading function (in eukaryotes it would be DNA pol ε/δ) removes mispaired nucleotides from the 3’ end of the growing DNA- ie goes into reverse to excise bad nucleotide (growing 5’ to 3’) function is key to maintain the accuracy of DNA synthesis via this proofreading capacity. Diagram of DNA Polymerase I: The 3’–>5' exonuclease activity of the enzyme allows the incorrect base pair to be excised (this activity is known as proofreading).  DNA Repair DNA DAMAGE DNA most susceptible to environmental damage Ionizing radiation, common chemicals, UV radiation and thermal energy from normal metabolism → spontaneous alteration (lesions) in DNA 10,000 bases/day