DNA Replication & Related Processes: E. coli Replication Fork Lecture Notes

DNA synthesis and replication: E. coli DNA polymerase I vs. polymerase III: Kornberg purified the first DNA polymerase from E. coli capable of synthesizing DNA in vitro, and named it DNA pol I However, E. coli DNA pol I mutants are viable! à therefore, DNA pol I cannot be the main...

DNA synthesis and replication: E. coli DNA polymerase I vs. polymerase III: Kornberg purified the first DNA polymerase from E. coli capable of synthesizing DNA in vitro, and named it DNA pol I However, E. coli DNA pol I mutants are viable! à therefore, DNA pol I cannot be the main replicative polymerase (i.e. the polymerase responsible for replicating the E. coli genome) Instead, the main replicative polymerase in E. coli = DNA polymerase III 50 DNA synthesis and replication: E. coli DNA polymerase III holoenzyme: = multi-subunit complex that has 17 subunits. Contains four sub-assemblies: 1. The core polymerase consisting of three subunits: a (the polymerase); e (the 3'–5' exonuclease); and q (the stimulator of the 3'–5' exonuclease). 2. The sliding clamp comprising two homodimers of the b subunit, which provides the ring structure that is needed for processivity. 3. The t subunit – responsible for dimerization of the core DNA polymerase. 4. Five subunits that have clamp-loader functions — g, d, d', y and c. Helicase 51 Components of DNA pol III holoenzyme: The E. coli b-clamp: Interacts with the a (polymerase) subunit of the pol III holoenzyme. Assembles into a dimer with a 35 Angstrom diameter hole in the middle. Confers extended processivity to the DNA pol III holoenzyme – can synthesize at least 1.5 x 105 bases of the leading strand without dissociation. Also increases rate of DNA synthesis (~ 750 - 1000 nts/s). 52 Components of DNA pol III holoenzyme: Clamp is needed to keep DNA pol III on DNA: Most polymerases only synthesize a short stretch of DNA before falling off. The b-ring dimer allows the DNA pol III holoenzyme to stay on DNA and be highly processive. The clamp guide the holoenzyme and slide along the DNA duplex, but not ss DNA. The b-rings are assembled onto DNA by the clamp loaders, and the assembly/disassembly of the b-rings require ATP as energy. 53 DNA synthesis and replication: Model for E. coli replication fork: Helicase unwinds dsDNA Note opposite orientations of leading and lagging strand polymerases, and both travel in same direction as replication fork 54 DNA synthesis and replication: Model for E. coli replication fork: New primer synthesized by primase 55 DNA synthesis and replication: Model for E. coli replication fork: Primase and polymerase are both released Primase is a distributive enzyme (i.e. comes off and reloads onto DNA through interaction with DnaB) 56 DNA synthesis and replication: Model for E. coli replication fork: 57 DNA synthesis and replication: Model for E. coli replication fork: b-ring with primed DNA strand is attached to lagging strand polymerase 58 DNA synthesis and replication: E. Coli replication fork: https://dnalc.cshl.edu/resources/3d/04-mechanism-of-replication- advanced.html 59 DNA synthesis and replication: E. Coli DNA pol III at replication fork: Model largely based on crystal structures of different components of the replication fork, and the necessity of replicating both the leading and lagging strand at the same time. Looping of the lagging strand is supported by electron micrograph (EM) pictures. 60 DNA replication and related processes: Cycle of loading and unloading DNA polymerase and clamp protein: On the leading strand, the moving DNA polymerase is tightly bound to the clamp and the two remain associated for a long time. On the lagging strand, each time the polymerase reaches the 5’ end of the proceeding Okazaki fragment, the polymerase is released, and this polymerase molecule then associates with a new clamp assembled on the RNA primer of the next Okazaki fragment. 61 DNA replication and related processes: DNA primase synthesizes RNA primers: DNA primase synthesizes RNA primers (~ 10 - 12 nt long) de novo to prime synthesis of Okazaki fragments. Primase is highly error-prone; however, synthesis of RNA primers does not require high fidelity since they eventually get removed 62 DNA replication and related processes: Removal of RNA primers: After synthesis of the Okazaki fragments, the RNA primers must be removed and replaced by DNA. This step is mediated by RNase H and DNA pol I. Ligase is needed to join Okazaki fragments. 63 DNA replication and related processes: DNA ligase seals the nicks on the lagging strand: Two classes of ligases: one uses NAD+ as cofactor (e.g E. coli), the other uses ATP as cofactor (eukaryotes). NAD = nicotinamide adenine dinucleotide NMN = nicotinamide mononucleotide NMN = nicotinamide mononucleotide 64 DNA replication and related processes: Termination of E. coli replication: The E. coli replication forks proceed in bi-directional manner until they into each other or when they hit the termination (Ter) sites. Ter is a short consensus DNA sequence (~ 23 bp) where the Tus (terminus utilization substance) protein bind. Tus is a 36 kD protein that binds DNA as a monomer. It has asymmetrical domains and its asymmetric binding to Ter enable the arrest of the replication fork progression in a directional manner. Why so many Ter sites and why this arrangment? 65 DNA replication and related processes: Termination of E. coli replication: When DnaB helicase runs into Tus-Ter from the permissive side, will displace Tus from Ter. When DnaB helicase runs into Tus-Ter from non-permissive side, will be trapped and locked. Eventually replication fork machinery disassembles (starting with the removal of the helicase) at the Tus-Ter sites. 66 DNA replication and related processes: Termination of E. coli replication: The Tus-Ter design only allows one way passage of the moving replication fork. 67 DNA replication and related processes: Fidelity of E. coli replication: Multiple factors help lower the error rate during DNA replication: This is roughly one mutation per 250 rounds of replication of the E. coli genome 68 DNA replication and related processes: Fidelity of E. coli replication: Genetics has been very useful for identifying components important for maintaining DNA replication fidelity (e.g. more Forward genetics screens): à Random mutation and isolate strains of E. coli (and other organisms) that have higher (mutator) or lower (anti-mutator) mutation rates. à Identify the genes that are mutated and study the causes of these phenotypes. Contribution of nucleotide selectivity, proofreading and DNA mismatch repair to DNA replication fidelity. The figure depicts the wide-ranging contributions of three major processes that act in series to determine DNA replication fidelity. The colored brackets illustrate the investment each process makes to the overall fidelity. Conditions that comprise each process are shown on the right 69 DNA replication and related processes: Fidelity of E. coli replication: The first proofreading step is carried out by the DNA polymerase and occurs before the new nucleotide is added to the growing chain. A) the correct nucleotide with the right geometry fit with the complementary base has higher affinity for the moving polymerase compared to the incorrect nucleotide. B) the active site of the polymerase only accommodates base pairs that have the “right” size. 70 DNA replication and related processes: Fidelity of E. coli replication: C) after nucleotide binding (but before formation of covalent bond), the polymerase undergoes an induced conformational change (closing of the “fingers”), and any incorrectly bound nucleotide is more likely to be rejected. 71 DNA replication and related processes: Fidelity of E. coli replication: Second proof-reading step mediated by the 3’ – 5’ exonuclease activity of polymerase à actively remove mis-incorporated nucleotide. 72 DNA replication and related processes: Fidelity of E. coli replication: Final proof-reading method: Strand-directed mismatch repair mechanism further lowers error rates in DNA replication. MutS detects distortion of the DNA helix and binds to a mismatched base pair. MutL binds the MutS-DNA complex and activates MutH at near-by methylated GATC to introduce a nick on the unmethylated strand of the DNA duplex. MutS/MutL scan the nearby DNA for a nick, and once detected, MutL recruits helicase to separate DNA strands and exonucleases to degrade the nicked strand all the way back pass the mismatch. DNA polymerase (pol III) comes in and fills in the gap using the methylated strand as template. 73  DNA replication and related processes: Fidelity of E. coli replication: Example of a biochemical assay for studying mismatch repair à incubate plasmid with mismatch within EcoR1 site in extracts from wild type or mutant E. coli strains and assay for repair based on sensitivity to EcoR1 cleavage. E.g.: EcoR1S = 5’GAATTC3’ EcoR1R = 3’CCTAAG5’ Biochemical assay for mismatch correction: substrate = covalently closed heteroduplex containing a mismatch within the EcoRI site. Methyl groups indicate the locations of the four GATC sites within the DNA. Cleavage of mismatch heteroduplexes with EcoRI and BamHI yields just the full- length linear BamHI product since the hybrid EcoRI site is resistant to cutting. Mismatch correction on the strand containing the mutant EcoRI sequence renders the site sensitive to cleavage, and now treatment with both restriction endonucleases yields two fragments as indicated. 74 DNA replication and related processes: Eukaryotic DNA replication: Replication of eukaryotic cells is more complex and less well characterized. Basic steps of DNA synthesis and properties of DNA polymerases are mostly conserved between prokaryotes and eukaryotes. However, eukaryotic cells à genomes are much larger, and are chromatinized. Also, chromosomes are linear, with lots of origins of replication. DNA replication is strictly confined to S phase of cell cycle. Rate of replication is much slower (~ 75 nts/s) compared to prokaryotes(~ 1000 nts/s) à likely due to chromatin. Most chromosomal replicons do not have termination region like that of E. coli. 75 DNA replication and related processes: Eukaryotic DNA replication: Eukaryotic cells have many origins of replication. In yeast, origins are well defined and also called ARS (autonomously replicating sequences). In higher eukaryotes, origins are not well defined and difficult to identify. In higher eukaryotes, it is clear that not all potential origins of replication fire at the same time during replication. Regulation of origin firing is complex and actively investigated by research labs still. 76  DNA replication and related processes: 1. The origin recognition complex (ORC) binds to Eukaryotic DNA replication: DNA and provides a site on the chromosome where additional replication factors can Early G1 associate. 2. Pre-replicative complex formation involves the association of Mcm2-7 complex with DNA at ORC. 3. Mcm2-7 proteins provide helicase activity for DNA synthesis and loading of these proteins confers competence on the origin to fire in S phase. 4. Onset of DNA synthesis requires the action of two protein kinases (cyclin dependent kinase S (CDK) and Cdc7), which trigger the association of additional proteins with the origin. During the process of initiation, DNA G2 polymerases are also recruited and DNA synthesis starts. 5. During replication, Mcm2-7 proteins move away from the origin and further assembly of Simplified summary of steps in eukaryotic DNA replication pre-replicative complexes is blocked. This ensures that origins can only fire a single time 77 per cell cycle. DNA replication and related processes: Eukaryotic DNA replication: Once origin of replication fires, events/proteins at the replication forks are similar to that of E. coli. Pol a = primase + another DNA polymerase activity. Single strand binding protein is called RPA (Replication protein A). PCNA = b ring of DNA pol III. Eukaryotic clamp loader is called RFC (Replication factor C), and is highly similar to the prokaryotic clamp loader complex. Recent findings showed that leading strand is synthesized by Pol e whereas lagging strand switches between Pol a 78 and Pol d. DNA replication and related processes: Eukaryotic DNA replication: Main eukaryotic polymerases (there are additional ones): For base excision repair 79 DNA replication and related processes: Eukaryotic DNA replication: Cycles of switching between DNA Pol a and Pol d on the lagging strand: 1. Pol a synthesizes RNA primer (10 – 12 nts) and then ~30 nts of DNA 2. RFC displaces Pol a and recruits PCNA with Pol d 3. PCNA clamps Pol d on DNA 4. Pol d elongates Okazaki fragment synthesis 80 DNA replication and related processes: Eukaryotic DNA replication: DNA polymerase switching and processing of an Okazaki fragment on the lagging strand. A.As the DNA helicase promotes unwinding at the replication fork, DNA pol e with RFC and PCNA synthesizes DNA on the leading strand. DNA pol α initiates synthesis on the lagging strand by generating an RNA primer (red segment) followed by a short segment of DNA. Then, RFC and PCNA load a second DNA polymerase (d) to continue synthesis of the Okazaki fragment. A.As DNA pol d approaches the downstream Okazaki fragment, cleavage by RNase H1 removes the initiator RNA primer leaving a single 5′-ribonucleotide. Then, FEN1/RTH1 removes the last 5′-ribonucleotide. The resulting nick is sealed by DNA ligase. 81 DNA replication and related processes: Comparison of prokaryotic and eukaryotic DNA replication proteins: Note that PCNA is made up of homo-trimer whereas b-clamp is made up of dimer 82 DNA replication and related processes: Eukaryotic DNA replication: Termination of DNA replication in eukaryotes – not well defined. End when replication forks run into each other? 83 DNA replication and related processes: DNA replication in the context of chromatin: Eukaryotic DNA replication is even more complicated given presence of chromatin and nucleosomes. Need to disassemble chromatin ahead of the replication fork, and re-assemble nucleosomes post DNA replicaiton. PCNA directly binds to chromatin remodeling complexes such as CAF-1. 84 DNA replication and related processes: Fidelity of DNA replication in eukaryotes: Basically same as in prokaryotes, however, larger selection of polymerases with different error rates. High fidelity polymerases Low fidelity polymerase for by passing DNA lesions 85 DNA replication and related processes: Eukaryotic mismatch repair: Again, similar to prokaryotic system except not methyl-directed. Initiated by nick on one strand à = repaired strand 86