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Alberts • Johnson • Lewis • Morgan • Raff • Roberts • Walter Molecular Biology of the Cell Sixth Edition Chapter 5 DNA Replication, Repair, and Recombination Copyright © Garland Science 2015 ”…short-term survival of cell can depend on preventing changes in its DNA, the long-term survival of a species requires that DNA sequences be changeable over many generations…” Topics • DNA replication mechanisms • The initiation and completion of DNA replication in chromosomes • DNA repair • Homologous recombination • Pages: 237-287 Terms to know • • • • • • • • • • DNA helicase DNA ligase DNA polymerase DNA primase DNA topoisomerase Lagging and leading strand Replication fork RNA primer Single strand DNA binding protein Strand directed mismatch repair Chemistry of DNA synthesis • Base pairing between incoming nucleotide and existing strand of DNA guides formation of new strand • Incoming nucleotide provides energy for reaction • What would happen if ddCTP were added to a DNA replication reaction in large excess over dCTP • What would happen if ddCTP were added to 10% of the concentration of dCTP? • What would happen if ddCMP were added to 10% of the concentration of dCTP or in large excess? Current view of the events at the bacterial replication fork Proofreading mechanisms of DNA replication • Overall fidelity of replication is about 1 in 1010 • Initial base-pairing is not sufficient to maintain fidelity of DNA replication – Besides the standard complementary base pairs also alternative base pairing is possible – With slight changes in helix geometry two H-bonds can be formed between G and T – Tautomeric forms of the four base pairs, e.g. C pairs with A, G with T DNA synthesis – effect of correct base pairing • Correct nucleotide has higher affinity for the moving polymerase as correct base pairing is energetically more favorable • Conformational change of polymerase occurs more readily with correct base pairing Exonucleolytic proofreading by DNA polymerase • C accidentally incorporated at the end of the growing end • For further elongation a basepaired 3´-OH end needs to be available, unpaired ends not readily extended • 3’ to 5’-exonucleolytic proofreading activity removes unpaired and also mispaired residues Strand distinction mechanisms • How to differentiate between newly synthesized and template DNA? • E. coli/prokaryotes – Methylation of A residue in the sequence GATC – Methylation occurs some time after DNA replication and allows distinguishing new from old strand • Eukaryotes: – Recognition is based on nicks on the DNA – On the lagging strand nicks are a result from discontinous DNA synthesis, leading strand nicks are introduced by unknown mechanism Strand-directed mismatch repair • Error rate after replication is about 1 error in 107 bases • Mismatch repair detects distortions in the DNA helix • MutS binds to mismatch • MutL scans for nick and triggers degradation of nicked strand • In bacteria MutH introduces nicks at unmethylated GATC Pro- and eukaryotic DNA replication • Fundamental features of pro- and eukaryotic DNA replication are conserved • More components in eukayotic replication machines, e.g. different polymerases for leading and lagging strand • Eukaryotic replication machinery must replicate through nucleosomes • Eukaryotic DNA replication tighly coordinated with mitosis Initiation of replication – a replication bubble formed by replication fork initiation • The position at which DNA is first opened is called replication origin • DNA around replication origin are normally rich in AT base pairs • Two replication forks are finally formed and move in opposite directions Initiation of DNA replication in bacteria • Binding of initiator proteins to DNA destabilizes DNA double helix • Helicases (DnaB) are brought in by Helicase loading proteins preventing binding to other ssDNA. • Helicase opens up the single stranded region enabling primases (DnaG) to bind • Completion of repliation fork DnaA DnaB DnaG +SSBproteins Control of replication initiation • Replication and cell divison are not necessarily synchronized but cell must ensure that only one round of replication occurs per cell cycle • Premature onset of next replication rounds must be prevented • Seq A protein binds to hemimethylated origin blocking initiator proteins • Origin becomes replication competent after methylation and dissociation of Seq A (in E.coli Dam methylase is responsible) Multiple origins of replication exist in mammals • Replication rate: 50 nucleotides per second • Average length of human chromosome: 150*106 bp – 30000 – 50000 origins of replication present on single chromosome DNA replication initiation in eukaryotes • Mechanism must ensure that each ori is only activated once per cell cycle • Prereplicative complex forms during G1 phase • Phosphorylation activates helicase and leads to displacement of origin recognition complex (ORC) • Replication takes places during S phase • ORC rebinds in phosporylated form and cannot accept helicase unless it becomes dephosphorlyated in G1 phase Chromosome structure and replication • Chromosome replication requires synthesis and assembly of new chromosomal proteins • Histone octamer broken into H3-H4 tetramer and H2A-H2B dimer • H3-H4 tetramer stays loosely attached to DNA, randomly distributed between both strands • Nucleosomes complemented with newly synthesized histone proteins • Lenght of Okazaki fragments restricted by positions of nucleosomes Displacement requires also chromatin remodeling complex Replication of DNA ends: Telomer replication • The final RNA primer on the lagging-strand template cannot be replaced by DNA because there is no free 3’-OH end available • Telomere DNA is recogized by sequence-specific DNA-binding proteins • Telomerases recognize tip of telomere DNA and elongates it in the 5’-3’ direction using an RNA template Terms to know • • • • • Base excision repair DNA repair Nonhomologous end joining Nucleotide excision repair Homologous recombination DNA repair • DNA is highly stable material • Susceptible under physiological conditions to spontaneous changes – Hydrolysis of N-glycosyl linkages to deoxyribose, depurination & depyrimidation – Deamination of cytosine – Encounter with reactive metabolites, oxygen, Sadenosylmethionine – UV-radiation • DNA is under constant surveillance for damage Deamination = hydrolysis methylation oxidation Hydrolysis Hydrolysis • These DNA lesions are result of normal chemical reactions that take place in a cell. • Exposure to external chemicals increases formation and greater variety modifications occurs Base excision repair • Mediated by set of DNA glycosylases, recognizing a specific type of altered base • Glycosylases move along DNA and flip bases out of the helix • Glycosylases probe all faces of the base and recognize modifications • Damaged base is removed Recognition of unusual nucleotide in DNA by base-flipping Nucleotide excision repair • Targets bulky lesions such as pyrimidine dimers, chemical modifications • Enzyme complex scans DNA for distortions in the helix • Phosphodiester backbone is cleaved on both side of lesion and single-stranded oligonucleotide is removed Repair of double-strand breaks • DNA double-helix breaks occur due to ionizing radiation, replication errors, oxidizing agents and other metabolites • Two distinct mechanisms exist – Nonhomologous end joining, quick and dirty solution by joining two ends by DNA ligase – Homologous recombination, accurate repair mechanism • Nonhomologous end joining creates deletion • Homologous recombination restores the original DNA sequence CRISPR/Cas9 based genome engineering – a case for cellular DNA repair Shao et al., Nature Protocols 9:2493–2512 (2014) Non-homologous end joining • Initial degradation of the broken DNA ends is important because nucleotides at the site of initial break are often damaged. • Central role is played by the Ku protein, a heterodimer that grasps broken chromosome ends • Often creates scars in the genome • Any two free ends can be joined, e.g. leading to chromosome rearrangments Homologous recombination • Heteroduplex formation is an essential feature of HR Probing for complementary bases • For HR to occur the broken DNA has to be broughy into proximity with the homologous, but unbroken DNA Homologous recombination • Basic steps: – Creating 3’ overhang – Strand exchange by complementary base pairing – DNA synthesis and ligation of nicks • Exact pathways of HR differ from one case to the next Critical step: Strand invasion by RecA • ATP-bound RecA binds single stranded DNA holding it in an elongated form (groups of nucleotide triplets separated) • Protein-DNA filaments binds to dsDNA and stretches it destabilizing the helix • Invading strand probes the DNA for complementary regions using triplets, if match, adjacent tripletes are probed • If extensive match is found, ATP is hydrolysed and RecA dissociates Spotlight: lnactivation of NHEJ improves genetic engineering PLoS ONE 7(6): e39720. doi:10.1371/journal.pone.0039720