DNA Replication Lecture Notes PDF

DNA replication Dr Yusra Siddiqui [email protected] Genetic information flow DNA replication DNA Transcriptio n RNA...

DNA replication Dr Yusra Siddiqui [email protected] Genetic information flow DNA replication DNA Transcriptio n RNA Translation Protein  DNA replication DNA needs to be replicated before cells divide When is DNA replicated? How is DNA replicated? How is DNA replicated so accurately?  Intended learning outcomes After the lecture (and lecture consolidation) you should be able to: Explain how a DNA double helix provides a template for its own replication Describe the experiment that revealed the semiconservative nature of DNA replication. Compare the direction in which replication forks move with the direction in which the new DNA strands are synthesized. Describe the problem created by a moving replication fork and explain how DNA topoisomerases relieve this difficulty. Explain how DNA polymerase contributes to the accuracy of DNA replication. Explain how the mismatch repair system recognizes and corrects replication  DNA replication “It has not escaped our attention that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material” Watson JD, Crick FHC (1953) Nature 171: 737-8 - semiconservative replication: Meselson and Stahl, 1958  DNA replication is semiconservative (Meselson and Stahl 1958) CsCl From Genetics, genes, genomes and evolution Amended from: LadyofHats/ Wikimedia Commons / Public Domain  DNA synthesis Enzyme (DNA polymerase) + Mg2+ dNTPs (dATP, dCTP, dGTP, dTTP) Single stranded template DNA Primer 3’-OH DNA synthesis 5’ to 3’ direction 5′ 3′-OH 3′ 3′ 5′  DNA Synthesis Complementary base pairing 5’ to 3’ direction Requires 3’-OH residue to extend from Breakage of phosphoanhydride bond of dNTP Formation of a phosphodiester bond Figure 5-3 Molecular Biology of the Cell (© Garland Science) The eukaryotic cell cycle S-phase: before cells can divide, DNA needs to replicate Chromosome state reflects the need to replicate and partition the genetic material prior to cell division Telomeres – areas of highly repetitive DNA that protect chromosome ends from DNA degradation, recombination, and end fusion with other chromosomes Centromeres - repetitive DNA which forms the spindle attachment site in mitosis Origin of replication - special sequence where duplication of the DNA begins; each chromosome will have G1 S G2 M G1 many origins  DNA replication is bidirectional from origins Replication bubbles in DNA visualised by electron microscopy. Essential Cell Biology, Fifth Edition Copyright © 2019 W. W. Norton & Company  Eukaryotic genomes vs Prokaryotic genomes Eukaryotic genomes are large and Bacterial genomes are small, compact arranged as linear chromosomes and (usually) circular. e.g. H. sapiens genome ~ 3000 million base pairs e.g. E. coli genome ~ 5 million base pairs Bacterial DNA has one origin of replication Theta (θ) structure Figure 5-6, 5-24 Molecular Biology of the Cell (© Garland Science)  Replication fork Both strands copied at replication fork Synthesis in 5’ to 3’ direction e h ?? t s ?? t i ? a h ere W h u e is s Essential Cell Biology, Fifth Edition Copyright © 2019 W. W. Norton & Company Replication fork Leading strand synthesis - continuous Lagging strand synthesis - discontinuous “Okasaki fragments” joined together Essential Cell Biology, Fifth Edition Copyright © 2019 W. W. Norton & Company DNA replication fork Leading strand synthesis - continuous Lagging strand synthesis - discontinuous “Okasaki fragments” joined together DNA replication fork Leading strand synthesis - continuous Lagging strand synthesis - discontinuous “Okasaki fragments” joined together  Lagging strand synthesis Essential Cell Biology, Fifth Edition Copyright © 2019 W. W. Norton & Company  DNA replication: lots of enzymes are working at the replication fork primase (RNA pol): Synthesise RNA primer + other factors including for example nucleases and DNA ligase Sliding clamps: Keep DNA pol. on the DNA (allow fast synthesis eg Eukaryotes ~50 bp s-1 at each fork and E. coli ~1000 bp s-1 Helicases: Unwind at each fork the DNA Single stranded DNA- binding proteins: Stabilise ssDNA DNA polymerases: Essential Cell Biology, Fifth Edition Synthesise new DNA Copyright © 2019 W. W. Norton & Company Lots of enzymes are working at the replication fork: act together as a replication machine. Essential Cell Biology, Fifth Edition Copyright © 2019 W. W. Norton & Company  Other activities associated with replication… Helicase unwinding of DNA causes supercoiling (“twists”) ahead of the replication fork which need to be unwound by topoisomerases Topoisomerases “untwist” DNA by breaking and re-forming phosphodiester bonds Essential Cell Biology, Fifth Edition Copyright © 2019 W. W. Norton & Company  The accuracy of DNA replication Replication step 5’3’ polymerization 1 in 105 (Errors per nucleotide added) 3’ 5’ exonucleolytic proofreading 1 in 102 (Probability of an error not being corrected) Strand-directed mismatch repair 1 in 103 (Probability of an error not being corrected) Combined 1 in 1010 Adapted from Alberts et al Molecular biology of the cell  5’3’ polymerization Replication step Errors per nucleotide added 5’3’ polymerization 1 in 105 Figure 5-4 Molecular Biology of the Cell (© Garland Science)  DNA polymerase can also “proofread” Figure 5-8 (part 2 of 2) Molecular Biology of the Cell (© Garland Science) Replication step 5’3’ polymerization 1 in 105 3’ 5’ exonucleolytic 1 in 102 (Errors not corrected) Essential Cell Biology, Fifth Edition proofreading Copyright © 2019 W. W. Norton & Company  Mismatch repair protein MutS detects incorrect base pairing in newly-synthesised DNA DNA kinks due to mismatched base pair: The MutS protein scans along the DNA looking for these kinks and recruits DNA repair proteins to them Replication step 5’3’ polymerization 1 in 105 (Errors per nucleotide added) 3’ 5’ exonucleolytic proofreading 1 in 102 (Errors not corrected) Strand-directed mismatch repair 1 in 103 (Errors not corrected)  Mismatch repair protein MutS detects incorrect base pairing in newly-synthesised DNA Mutations in human Mismatch Repair genes are associated with predisposition to some cancers, eg mutations in MutS are associated with a type of colon cancer Replication step 5’3’ polymerization 1 in 105 (Errors per nucleotide added) 3’ 5’ exonucleolytic proofreading 1 in 102 (Errors not corrected) Strand-directed mismatch repair 1 in 103 (Errors not corrected) Combined 1 in 1010 Table 5-1 Molecular Biology of the Cell (© Garland Science)  Key take home messages DNA replication occurs in S-phase, and is semi-conservative DNA polymerase is the key enzyme involved in DNA synthesis DNA synthesis proceeds 5’ to 3’, and is semi-discontinuous DNA replication requires coordination of multiple enzymes into a “replication machine” and at the replication fork and beyond. DNA replication is highly accurate due to DNA polymerase accuracy and proofreading, and DNA repair mechanisms  Recommended reading material: Relevant areas of one of the core textbooks or other general genetics textbook – for example… Essential cell biology Chapter 6 –DNA replication and repair iGenetics Chapter 3 – DNA Replication DNA replication video: http://www.dnalc.org/resources/3d/04-mechanism-of-replication-advanced.html Nobel prize page for DNA mismatch repair gives an overview of the process: http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2015/