Lecture 5: DNA Replication I

DNA replication Side notes: Replication is semi-conservative DNA replication is the complete, faithful copying of the DNA comprising the cell's chromosomes Replication is semi-conservative - each strand of the parent double helix acts as a template for synthesis of a new daughter DNA strand Each DNA strand serves as a template for synthesis of a new complementary To copy the template strand: The base on the template strand must be identified The complementary base must be added (A with T, C with G) This ensures that (barring mutations) each daughter double-helix is identical to the parent double-helix, and each daughter cell will receive identical DNA molecules dNTP additions proceed via nucleophilic attack Adding nucleotides to DNA requires energy Cells couple that unfavorable reaction with an energy generating reaction Hydrolysis of a nucleotide triphosphate provides nucleotide monophosphate and energy, both needed for the reaction NTPs are hydrolyzed to nucleotide monophosphates and pyrophosphate Pyrophosphate hydrolysis to monophosphates releases additional energy and is coupled to bond formation Replication initiates at origins of replication Replication starts at origins of replication Replication moves away from the origin in both directions forming a replication bubble The DNA double-helix is opened at the origin of replication and unwound to form replication forks At replication forks, single stranded DNA (ssDNA) is exposed and DNA synthesis can occur DNA synthesis on both strands occurs in a 5' to 3' direction This means that synthesis is continuous on one strand (the leading strand), but on the other strand (lagging strand) it is discontinuous Structure of DNA polymerase The structure of DNA polymerase resembles a right hand, with three domains called the thumb, fingers, and palm The single-stranded DNA is fed past the fingers and into the active site in the palm, where nucleotide addition is catalyzed The finger domain helps position the incoming nucleotide, which joins to the free 3' OH in the polymerase active site in the palm The thumb domain holds the elongating dsDNA Polymerase active site catalyzes addition of nucleotides The active site of DNA polymerase (in the palm domain) has carboxylate groups of two aspartate residues coordinated with two magnesium sons The active site catalyzes a phosphoryl transfer reaction linking the 5' phosphate of the incoming nucleotide to the 3' OH of the growing DNA to form a phosphodiester bond One of the Mg2+ ions activates the 3' OH while the second Mg2+ interacts with the incoming dNTP DNA replication Side notes: Initiation = opening, helicase loading, primer addition The origin of replication is recognized by an initiator protein that opens up the double helix and recruits helicases DNA helicases unwind the helix to expose single-stranded DNA, which is coated with ssDNA binding proteins DNA synthesis needs a primer - it can only add nucleotides to an existing 3' end The primer is a short RNA strand synthesized by primase Elongation = sliding clamp & DNA polymerase loading After the RNA primer is synthesized, the sliding clamp is recruited DNA polymerase is associated with DNA via the clamp loader and the sliding clamp The replication machinery moves along the DNA, copying the strands Each base in the parental DNA is read by DNA polymerase, which adds complementary bases to the growing strand in a 5' to 3' direction Termination = RNA primer removal, DNA ligation Termination occurs when two different forks meet, when the fork reaches the end of a linear chromosome, or when polymerase meets the previously replicated strand Replication complexes are disassembled RNA primers are removed and replaced with DNA DNA ligase connects adjacent strands Initiation of replication in bacteria In bacteria, the inflator protein is DNA A DNA A binds to specific sequences adjacent to origins of replication DNA A binding allows hellcats to bind to an upstream AT-rich region The helicases unwind the DNA, allowing replication machinery to bind Bacteria: DNAA unwinds DNA at origin dsDNA must be actively unwound to provide access to ssDNA for replication In E. coli the origin (OriC) has a 245 bp sequence, with seven 9 bp DNAA boxes that bind DNAA When bound to ATP, the AAA + domains of DNAA multimerize into a spiral filament The filament interaction with DNA at the origin distorts the DNA. Which facilitates unwinding at the adjacent AT rich region Bacteria: DNAA helps load helicase As well as generating ssDNA at the DNAA also recruits DNAB helicase to the origin Helicases unwind DNA DNAB (helicase) is loaded onto the DNA by DNAC (helicase loader) Once the helicase is loaded, primase and polymerase can be recruited and replication can begin The ORC dissociates once the MCM2-7 pair is loaded Initiation of replication in eukaryotes In eukaryotes, the origin recognition complex (orc) is the initiator, and has 6 subunits In most eukaryotes, the orc binds the DNA in a sequence-independent manner The orc recruits Cdc6 and Cdt1 proteins These sequentially load two ring-shaped MCM2-7 hexamers in a head-to-head orientation DNA Replication Side notes: Eukaryotes: helicase complex unwinds DNA The MCM2-7 pair is then activated by accessory proteins such as Cdc45 Other proteins are loaded, including polymerase, and then the full helicase complex (called CMG) can be activated by phosphorylation to unwind DNA and start replication Bacterial and eukaryotic helicases work differently Helicases in bacteria and eukaryotes are similar - they are both AAA + proteins and have 6 subunits in a ring structure However, DNAB (bacteria) and MCM (eukaryotes) have very different structural folds and they work differently Also, bacterial helicase moves along the lagging strand in a 5' to 3' direction and eukaryotic helicase moves along the leading strand in a 3' to 5' direction These features suggest bacterial and eukaryotic helicases evolved independently Top hat l In bacteria, DNAA binds and _______ DNA at the origin of replication Unwinds Top hat 2 This protein complex is the _______ Helicases Single-stranded binding proteins stabilize DNA Single stranded DNA can form secondary structures that would make copying by polymerase difficult Single-stranded binding proteins bind to the displaced single-stranded DNA Bacteria = SSB Eukaryotes = replication protein A (RPA) Primase synthesizes RNA primers that start new DNA strands DNA polymerase requires existing 3' OH for synthesis RNA primers must be synthesized on the leading strand (infrequently) and lagging strand (frequently) Primase binds to ssDNA coated with ssDNA binding proteins Primase does not require an existing 3' OH, and synthesizes ~10 nucleotides of RNA In eukaryotes, polymerase alpha subunit of primase synthesizes short piece of DNA before handing over to replicative polymerases (polymerase switching) The sliding clamp tethers DNA polymerase to DNA After the synthesis of the primer, replicative DNA polymerase must be brought to the DNA - this is done by interaction with the sliding clamp, which is loaded at the template-primer junction by clamp loader The clamp and clamp loader are highly conserved in bacteria, archaea and eukaryotes Bacterial sliding clamp = beta protein Eukaryotic sliding clamp = PCNA The high processivity of replicative DNA polymerase is due to the sliding clamp that keeps it tethered to DNA The sliding clamp loader binds and opens sliding clamp ring The sliding clamp is a ring, so it must be opened before it can fit around DNA this is done by the clamp loader The clamp loader is a 5-subunit ring structure (replication factor C; RFC in eukaryotes) Some clamp loader subunits are AAA+ ATPases - when ATP binds, the conformational change drives clamp loading On binding ATP, the clamp loader binds to the sliding clamp, forming a spiral shape The clamp loader-sliding clamp complex has a high affinity with the 3' end of DNA Replication Side notes: the primer-template junction Binding of the complex to the primer-template junction stimulates ATPase activity, which closes the sliding clamp and releases the clamp loader A polymerase switch occurs in eukaryotes In eukaryotes, after the polymerase alpha submit of the primase complex makes a short stretch of DNA, replicative polymerases take over Polymerase alpha is replaced by replicative polymerase (on the lagging strand) or epsilon (leading strand) Dissociation of polymerase alpha exposes the primer template junction and recruits the clamp loader-sliding clamp complex The clamp loader-sliding clamp then recruits polymerase - this ensures that replicative polymerases only bind when a primer has been synthesized Establishing replication fork in bacteria Establishing the replication fork is different in bacteria and eukaryotes In bacteria, DNAA binds to oriC and opens the helix Helicase loader DNAC assembles with DNAB helicase and loads directly onto SSB coated single-stranded DNA DNAB recruits the primase DNAG, which initiates strand synthesis The clamp loader-sliding clamp complex binds to the primer-template junction After the loader dissociates, the sliding clamp recruits replicative DNA polymerase III Top hat 3 This protein complex is the _________ Sliding clamp Establishing replication fork in eukaryotes In eukaryotes, the orc complex binds to DNA and loads the core helicase-MCM complex onto double-stranded in a head-to-head fashion Helicase is then activated, DNA unwound, and single-stranded DNA bound by RPA Additional subunits bind to MCM to make the active CMG helicase complex Other components, including replicative DNA polymerase epsilon, associate with CMG, and the help is then opened up Polymerase alpha-primase initiates synthesis, then polymerase switching occurs and polymerase epsilon is recruited to the leading strand by PCNA Polymerase alpha-primase initiates synthesis on the lagging strand, followed by another polymerase switch to polymerase How is replication fidelity ensured? Overall error rate of DNA replication is 1 in every 10,000,000,000 nucleotides Nucleotide selection by DNA polymerase (i.e. "Fit") Proofreading and exonucleolytic removal by polymerase Mismatch repair after replication Nucleotide selection by DNA polymerase Replicative polymerases only make one error per ~100,000 nucleotides During addition, the polymerase recognizes the correct nucleotide due to the precise fit in the active site when base-paired with the template strand Mismatches have a different shape to the correct bases and don't fit in the active site as well No energy is required for this level of control Proofreading provides a second layer of fidelity Proofreading is achieved by 3' to 5' exonuclease activity, where an incorrect nucleotide is removed before the next nucleotide is added DNA Replication Side notes: The polymerase and exonuclease active sites are spatially separated - if no error is detected, the polymerase moves to the next nucleotide addition If an incorrect nucleotide is detected, the incorporation rate slows to allow time for the error to be corrected The end of the newly-synthesized strand, with the incorrect nucleotide, is moved to the exonuclease site and removed This proofreading step - which requires energy - provides an extra 100-fold level of fidelity, bring the error rate at this stage down to 1 in 10^7 nucleotides