Lecture 7 DNA Replication PDF

Lecture 7 DNA replication Additional material for this lecture may be found in: § Lehninger’s Biochemistry (8th ed), chapter 25: p. 914-930 WHAT IS DNA METABOLISM ? Although DNA provides stable storage of genetic information, the structure is far from static: – A new copy of DNA is synthesized with...

Lecture 7 DNA replication Additional material for this lecture may be found in: § Lehninger’s Biochemistry (8th ed), chapter 25: p. 914-930 WHAT IS DNA METABOLISM ? Although DNA provides stable storage of genetic information, the structure is far from static: – A new copy of DNA is synthesized with high fidelity before each cell division (DNA replication) – Errors that arise during or after DNA synthesis are constantly checked for, and repairs are made (DNA repair) – Segments of DNA are rearranged either within one DNA (chromosome) or between two DNA molecules, (DNA recombination) giving offspring a novel DNA DNA metabolism consists of a set of tightly regulated processes that achieve these tasks DNA is the substrate that encodes its own metabolism DNA METABOLISM 1 Key topics: –DNA replication in bacteria –DNA replication in eukaryotes REPLICATION IN BACTERIA DNA ELONGATION CHEMISTRY Elongation of a DNA chain: (dNMP)n + dNTP à (dNMP)n+1 + PPi DNA à Lengthened DNA Parental DNA strand serves as a template Nucleoside triphosphates (dNTPs) serve as substrates in strand synthesis The nucleophilic OH group at the 3’ end of growing chain (the primer) attacks the a-phosphate of the incoming trinucleotide – This 3’-OH is REQUIRED – The 3’-OH is made a more powerful nucleophile by nearby Mg2+ ions Pyrophosphate (made of the b and g phosphates; PPi) is a good leaving group MECHANISM OF ACTION OF DNA POLYMERASES Requirement for DNA Polymerase: A template and a primer (and dNTPs) The catalytic mechanism for addition of a new nucleotide by DNA polymerase involves two Mg2+ ions, coordinated to the phosphate groups of the incoming nucleotide triphosphate, the 3’-hydroxyl group that will act as a nucleophile, and three Asp residues, two of which are highly conserved in all DNA polymerases. The Mg2+ ion depicted at the top facilitates attack of the 3’-hydroxyl group of the primer on the α phosphate of the nucleotide triphosphate; the other Mg2+ ion facilitates displacement of the pyrophosphate. Both ions stabilize the structure of the pentacovalent transition state. RNA polymerases use a similar mechanism An important feature of DNA Polymerase: Processivity (number of dNTPs added before it dissociates from the template) FEATURES AND IMPORTANCE OF A PRIMER Primer = short strand complementary to the template – Contains a free 3’-OH to begin the first DNA polymerasecatalyzed reaction – Can be made of DNA or RNA § Each incoming nucleotide is selected in part by base-pairing to the appropriate nucleotide in the template strand. The insertion reaction occurs in the insertion site § The reaction product has a new free 3’ hydroxyl, allowing the addition of another nucleotide. § The newly formed base pair migrates to the post-insertion site to make the active site available to the next pair to be formed. FEATURES OF DNA POLYMERASE DNA POLYMERASE has a pocket with two regions: – Insertion site: where the incoming nucleotide binds – Post-insertion site: where the newly made base pair resides when the polymerase moves forward The core of most DNA polymerases is shaped like a human hand that wraps around the active site. The structure shown is the DNA polymerase I of Thermus aquaticus, bound to DNA. The cartoon interpretation of the polymerase structure shows the insertion and postinsertion parts of the active site. The insertion site is where the nucleotide addition occurs, and the postinsertion site is where the newly formed base pair migrates after it appears. DNA POLYMERASE CAN ADD NUCLEOTIDES OR DISSOCIATE The number of nucleotides added before dissociation is called processivity Polymerases have widely varying processivity rates GEOMETRY OF BASE PAIRING AND PROOFREADING ACCOUNTS FOR HIGH FIDELITY In principle, DNA polymerase active site excludes base pairs with incorrect geometry, and it also has a proofreading activity. – BUT DNA polymerases still insert wrong base every 1/104–1/105 (because of tautomerism à H-bonding to incorrect partner) – And proofreading (see next slides) still does not correct errors every 1/102 bases – Thus, the error ratio is still ~ 1/106 to 1/107 bases – Repair mechanisms fix these errors with an error rate of 1/103 bases Thus, the combined error rate in E. coli is: 1/109 – 1/1010 bases – (since E. coli chromosome is ~ 4,6 106 bp, errors occur at a ratio of 1 per 1000 – 10,000 replications) HIGH FIDELITY OF DNA SYNTHESIS CONTRIBUTION OF BASE PAIR GEOMETRY TO THE FIDELITY OF DNA REPLICATION (a)The standard A=T and G≡C base pairs have very similar geometries, and an active site sized to fit one will generally accommodate the other. (b)The geometry of incorrectly paired bases can exclude them from the active site, as occurs on DNA polymerase. ERRORS DURING SYNTHESIS ARE CORRECTED BY 3‘à5’ EXONUCLEASE ACTIVITY (PROOFREADING) Almost all DNA polymerases have an additional activity: 3’à5’-exonuclease activity “proofreads” synthesis for mismatched base pair Translocation of enzyme to next position is inhibited until the enzyme can remove the incorrect nucleotide ERROR CORRECTION (PROOFREADING) BY 3’à5’-EXONUCLEASE ACTIVITY OF DNA POLYMERASE I § The exonuclease activity is located behind the polymerase activity as the enzyme is oriented in its movement along the DNA. § A mismatched base (here, a C–T mismatch) impedes translocation of DNA polymerase I to the next site. § The DNA bound to the enzyme slides backward into the exonuclease site, and the enzyme corrects the mistake with its 3’Ž5’ exonuclease activity. § The enzyme then resumes its polymerase activity in the 5’Ž3’ direction. § Proofreading activity of Pol I improves the accuracy of polymerization by 102 to 103 fold THERE ARE AT LEAST FIVE DNA POLYMERASES IN E. COLI DNA Polymerase I is abundant but is not ideal for replication: It has – A slow rate: 10-20 nucleotides/sec, which is slower than observed for replication fork movement – A low processivity (3-200 bases added before it dissociates à therefore, Its primary function is in clean-up DNA Polymerase III is the principal replication polymerase DNA Polymerases II, IV, and V are involved in DNA repair COMPARISON OF THREE DNA POLYMERASES OF E. COLI Dna Polymerases II, IV and V are involved in DNA repair DNA POLYMERASE I HAS ALSO A 5’à3’-EXONUCLEASE ACTIVITY (NICK TRANSLATION) In addition to the 3’à5’-exonuclease activity, DNA Polymerase I Degrades the DNA ahead of the enzyme, thus removing nucleotides on its path Thus, does nick translation―a strand break moves along with enzyme The Klenow fragment―a distinct domain that can be separated from DNA Polymerase I by protease treatment in the laboratory―comprises the 5’ to 3’ polymerase activity and the 3’ to 5’ proofreading exonuclease activity BUT NOT the 5’ to 3’ (exonuclease) nick translation activity. It is used as a tool for several applications in research. NICK TRANSLATION The bacterial DNA polymerase I has three domains, catalyzing its DNA polymerase, 5’Ž3’ exonuclease, and 3’Ž5’ exonuclease activities. The 5’Ž3’ exonuclease domain is in front of the enzyme as it moves along the DNA. By degrading the DNA strand ahead of the enzyme and synthesizing a new strand behind, DNA polymerase I can promote a reaction called nick translation, where a break or nick in the DNA is effectively moved along with the enzyme. Nick translation has a role in DNA repair and in the removal of RNA primers during replication (both described later). The strand of nucleic acid to be removed (either DNA or RNA) is shown in purple, the replacement strand in red. DNA synthesis begins at a nick (a broken phosphodiester bond, leaving a free 3’ hydroxyl and a free 5’ phosphate). A nick remains where DNA polymerase I eventually dissociates, and the nick is later sealed by another enzyme. THE REPLICATING POLYMERASE, DNA POLYMERASE III IS A COMPLEX STRUCTURE WITH 10 TYPES OF SUBUNITS Three core domains made of a, e, and q subunits The core domains are linked by the “clamploader” complex t3gdd’ The core domains each interact with a dimer of b subunits that increases the processivity of the complex – Form a sliding clamp that prevents dissociation – Processivity of DNA Pol III is >500,000 bp SUBUNITS OF DNA POLYMERASE III OF E. COLI DNA POLYMERASE III The circular clamp (2 subunits) surrounding the DNA DNA polymerase III. (a)Architecture of bacterial DNA polymerase III (Pol III). Three core domains, composed of subunits α, ε, and θ, are linked by a five-subunit clamp-loading complex (also known as the γ complex) with the composition τ3γδδ’. The core subunits and clamp-loader complex constitute DNA polymerase III*. The other two subunits of DNA polymerase III*, χ and ψ (not shown), also bind to the clamp-loading complex. Three β clamps interact with the three-core subassembly, each clamp a dimer of the b subunit. The complex interacts with the DnaB helicase (described later) through the τ subunits. (b)Two β subunits of E. coli polymerase III form a circular clamp that surrounds the DNA. The clamp slides along the DNA molecule, increasing the processivity of the polymerase III holoenzyme to greater than 500,000 nucleotides by preventing its dissociation from the DNA. The two b subunits are shown in two shades of purple as ribbon structures (left) and surface contour images (right), surrounding the DNA. E. COLI REQUIRES OVER 20 ENZYMES AND PROTEINS FOR DNA REPLICATION Replication of E. coli chromosome proceeds in stages (initiation, elongation termination) A set of proteins involved in replication, called the replisome, includes: – DNA-binding proteins to stabilize separated strands – Helicases (use ATP to unwind DNA strands) – Primases (make RNA primer) – DNA Polymerases I And III (primer removal and DNA elongation) – DNA ligases to seal nicks – DNA Gyrases (also called DNA Topoisomerases II: relieve the stress caused by unwinding) PROTEINS OF E. COLI REPLISOME FIRST PHASE OF REPLICATION: INITIATION Initiation of replication in E. Coli begins at Origin oriC – – – – oriC is 245 bp has highly conserved sequence elements Contains A=T-rich DNA Unwinding Element (DUE) Contains five repeats of a 9-bp sequence (R sites) that form binding site for initiator protein DnaA – with more binding sites for: DnaA (I sites), IHF (integration host factor), and FIS (factor for inversion stimulation) ARRANGEMENT OF CONSERVED SEQUENCES OF ORIC DUE: DNA Unwinding Element Dna A: Initiatior protein Consensus sequences for key repeated elements are shown. N represents any of the four nucleotides. The horizontal arrows indicate the orientations of the nucleotide sequences (left-to-right arrow denotes sequence in top strand; right-to-left, bottom strand). - FIS and IHF are binding sites for FIS and IHF proteins that activate initiation. R sites are bound by DnaA. I sites are additional Dna A-binding sites (with different sequences), bound by DnaA only when the protein is complexed with ATP. INITIATION REQUIRES AT LEAST 10 DIFFERENT PROTEINS Goal of the initiation phase: to open the DNA helix and to form pre-priming complex TABLE 25-3 Proteins Required to Initiate Replication at the E. coli Origin Mr Number of subunits DnaA protein 52,000 1 Recognizes oriC sequence; opens duplex at specific sites in origin DnaB protein (helicase) 300,000 6a Unwinds DNA DnaC protein 174,000 6a Required for DnaB binding at origin HU 19,000 2 Histone-like protein; DNA-binding protein; stimulates initiation FIS 22,500 2a DNA-binding protein; stimulates initiation IHF 22,000 2 DNA-binding protein; stimulates initiation Primase (DnaG protein) 60,000 1 Synthesizes RNA primers Single-stranded DNA-binding protein (SSB) 75,000 4a Binds single-stranded DNA DNA gyrase (DNA topoisomerase II) 400,000 4 Relieves torsional strain generated by DNA unwinding Dam methylase 32,000 1 Methylates (5')GATC sequences at oriC Protein aSubunits in these cases are identical. Function Dna A PROTEINS BIND AT R AND I SITES IN ORIC DnaA proteins are ATPases 8 DnaAs bind to R and I sites DNA wraps around the 8 DnaAs complex forming a supercoil (a helix of a helicoidal DNA: a superhelix) Strain in binding leads to denaturation (separation of the two DNA strands:“melting”) of nearby DUE sites DnaB HELICASE CONTINUES INITIATION Another protein, DnaB Helicase (hexamer ring structure) is opened by the binding of DnaC DnaB Helicase then binds to separated strands in the melted DUE DnaB Helicase migrates along ssDNA 5’à3’and unwinds the helix in preparation of DNA synthesis Other proteins (DNA Pol III, etc.) link to DnaB Single-stranded DNA-binding protein (SSB) stabilizes separated strands DNA gyrase relieves topological stress ahead of the replication forks (because DNA is circular in E. coli) INITIATION OF REPLICATION AT ORIC Model for initiation of replication at the E. coli origin, oriC. Eight DnaA protein molecules, each with a bound ATP, bind at the R and I sites in the origin. The DNA is wrapped around this complex, which forms a right-handed helical structure. The A=T-rich DUE region is denatured as a result of the strain imparted by the adjacent DnaA binding. Formation of the helical DnaA complex is facilitated by the proteins HU, IHF, and FIS, (not shown here because their detailed structural roles have not yet been defined). Hexamers of the DnaB Helicase bind to each strand, with the aid of DnaC protein. The DnaB helicase activity further unwinds the DNA in preparation for priming and DNA synthesis. DnaA HTH (DnaA Helix-Turn-Helix motif) INITIATION IS REGULATED TO OCCUR ONCE PER CELL CYCLE THROUGH BINDING OF Hda Hda (Hda stands for Homologous to DnaA) – binds to b subunits of DNA Pol III – stimulates hydrolysis of DnaA’s ATP DnaA complex then dissociates ADP dissociates ATP rebinds to DnaA to stimulate all over again Time scale: 20–40 mins REGULATION OF REPLICATION INITIATION VIA METHYLATION After replication, oriC sequence is hemi-methylated Hemi-methylated oriC sequences interact with plasma membrane (uses protein SeqA) After a period, SeqA dissociates, oriC sequences are released from membrane Dam methylase fully methylates DNA to allow new DnaA to bind – Dam = DNA adenine methylase – Methylates N6 of A in GATC sequences SECOND PHASE OF REPLICATION: ELONGATION 1/ Leading Strand Synthesis Primase (DnaG) makes RNA primer (5’ à 3’) – The DnaG primase interacts first with DnaB helicase (moves on the ssDNA in 5’ to 3’ direction), then primase moves in opposite direction to helicase to make the primer DNA Pol III adds nucleotides – Pol III is linked to DnaB, which is tethered to the opposite DNA strand 2/ lagging strand synthesis Synthesis of Okazaki fragments. (a) At intervals, primase synthesizes an RNA primer for a new Okazaki fragment. Note that if we consider the two template strands as lying side by side, lagging strand synthesis formally proceeds in the opposite direction from fork movement. Each primer is extended by DNA polymerase III. DNA synthesis continues until the fragment extends as far as the primer of the previously added Okazaki fragment. A new primer is synthesized near the replication fork to begin the process again. (b) In the replisome complex, DNA synthesis on the leading and lagging strands is tightly coordinated. Each DNA polymerase III holoenzyme has three sets of core subunits (yellow), linked together with a single clamp-loading complex, so one or two Okazaki fragments can be synthesized simultaneously, along with the leading strand. HOW THE TWO DNA STRANDS CAN BE ELONGATED IN OPPOSITE DIRECTION BY A SINGLE POLYMERASE III ? For the lagging strand, primase makes RNA primer and DNA Polymerase III adds nucleotides (as in leading strand synthesis) BUT the SAME DNA Polymerase III works on both strands! – How the 2 strands can be elongated in opposite direction (leading vs lagging strand) by a single DNA Polymerase III that moves in one direction? The DNA of the lagging strand loops around HOW A SINGLE POLYMERASE III CAN ELONGATE DIFFERENT, SUCCESSIVE OKAZAKI FRAGMENTS ? § DNA polymerase III holoenzyme is a dimeric polymerase § One unit of the polymerase III dimer, synthesizes the leading strand while the other synthesizes the lagging strand (see DNA looping : because DNA synthesis always proceeds in the 5’à3’ direction, as the template strand is read in the 3’à5’ direction, lagging strand synthesis must take place on a looped out template). § Lagging strand synthesis requires repeating priming by Primase bound to DnaB Helicase § All single strand regions of DNA are coated with SSB protein. From Garett & Grisham, 4th Ed. § DNA Polymerase III, moving in one direction can elongate different, successive, Okazaki fragments by transitioning between fragments TRANSITIONING BETWEEN OKAZAKI FRAGMENTS Core subunits of DNA Pol III dissociate from one b clamp then bind to a new one (brought by the clamp loader), starting elongation at a new primer The old b clamp is left behind ======================================= Once the two Okazaki fragments are close to each other RNA primer is removed by DNA Polymerase I DNA Polymerase I fills in the gap by nick translation DNA ligase seals the nick FINAL STEPS IN THE SYNTHESIS OF THE LAGGING STRAND § RNA primers in the lagging strand are removed by the 5’Ž3’ exonuclease activity of DNA polymerase I and are replaced with DNA by the same enzyme. § The remaining nick is sealed by DNA ligase which makes a bond between a 3’-OH and a 5’-PO4. - 5’-PO4 must be activated by attachment of AMP - 3’-OH nucleophile attacks this phosphate, displacing AMP GENERAL FEATURES OF THE REPLICATION FORK The DNA duplex is unwound by DNA Gyrase and Helicase Single strand regions are coated by SSB Primase periodically adds primers The 2 Sliding clamp of DNA polymerase III replicate the strands DNA polymerase I and ligase act downstream on the lagging strand to Remove DNA primers and replace them with DNA (Pol I) Ligate the Okazaki fragments (Ligase) From Garett & Grisham, 4th Ed. THIRD PHASE OF REPLICATION: TERMINATION Replication forks meet at region with 20-bp sequences Ter (TerA-TerF) – Ter sites are found near each other but in opposite directions – Create a site that replication forks cannot leave Ter is also a binding site for protein Tus – Tus = Terminus Utilization Sequence – Causes a replication fork to stop TERMINATION OF REPLICATION IN E. COLI § The Ter sequences (TerA through TerF) are positioned on the chromosome in two clusters with opposite orientations, and function as a trap for the replication fork. § Ter sequences bind Tus proteins (Terminus Utilisation Sequence) Topoisomerases are needed in replication termination Replication of the DNA separating opposing replication forks leaves the completed chromosomes joined as catenanes, or topologically interlinked circles. The circles are not covalently linked, but because they are interwound and each is covalently closed, they cannot be separated— except by the action of topoisomerases. In E. coli, a type II topoisomerase known as DNA topoisomerase IV plays the primary role in the separation of catenated chromosomes, transiently breaking both DNA strands of one chromosome and allowing the other chromosome to pass through the break. REPLICATION IN EUKARYOTES REPLICATION IN EUKARYOTES IS MORE COMPLEX THAN IN BACTERIA Yeast have ~400 well-defined origins – Called Autonomously Replicating Sequences (ARS) or replicators Entire genome replicated 1time/cycle – Regulation due to cyclin proteins and cyclindependent kinases (CDKs) – Cyclins are ubiquinated for proteolytic destruction at the end of the M (mitosis) phase INITIATION OF REPLICATION REQUIRES A PRE-REPLICATIVE COMPLEX The Origin Recognition Complex (ORC) protein binds to the replication origin then loads a helicase onto the DNA – ORC functions like bacterial DnaA/DnaC The Helicase is a hexamer of Mini-Chromosome Maintenance proteins (MCM2-7) – MCM2-7 function like bacterial DnaB helicase ASSEMBLY OF A PRE-REPLICATIVE COMPLEX AT A EUKARYOTIC ORIGIN u The initiation site (origin) is bound by ORC, CDC6, and CDT1. u These proteins, many of them AAA+ ATPases, promote loading of the replicative helicase, MCM2–7, in a reaction that is analogous to the loading of the bacterial DnaB helicase by DnaC protein. u Loading of the MCM helicase complex onto the DNA forms the pre-replicative complex, or pre-RC, and is the key step in the initiation of replication. LIKE BACTERIA, EUKARYOTES HAVE MULTIPLE DNA POLYMERASES – DNA Pol a probably used to make primers for Okasaki fragments. Has primase activity but no 3’à5’-proofreading activity – DNA Pol b probably used in DNA repair – DNA Pol g Mitochondrial DNA replication – DNA Pol d used in leading and Okazaki synthesis Comparable to bacterial DNA Pol III Has 3’à5’- proofreading activity – DNA Pol e used in DNA repair OTHER PROTEINS THAT PARTICIPATE TO REPLICATION IN EUKARYOTES – RPA: Single-Strand Binding Protein (equivalent to E. coli SSB) – RFC: Clamp loader for the polymerase (equivalent to E. coli clamp loader) – Telomerase: used in the synthesis of telomeres found at the end of linear chromosomes EUKARYOTIC REPLICATION OCCURS SLOWLY, BUT FROM MANY ORIGINS § Synthesis ~50 nucleotides/sec (~1/20th the rate seen in E. coli). § Synthesis is slow, but compensated by several replication origins (every 30–300 kilobases)