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Eukaryotic chromosomes are linear not circular - multiple origins of replication, not just one # DNA must be unwound # nucleosomes disaggregate and separate from DNA Eukaryotic DNA Replication Starts at the origin of replication # up to 60 origins /chromosome # 1 origin / 300kilobases Mechanism of initiation # Initiator proteins (in order of binding) Origin Recognition Complex (ORC) multisubunit protein complex bind to replication
Eukaryotic chromosomes are linear not circular - multiple origins of replication, not just one # DNA must be unwound # nucleosomes disaggregate and separate from DNA Eukaryotic DNA Replication Starts at the origin of replication # up to 60 origins /chromosome # 1 origin / 300kilobases Mechanism of initiation # Initiator proteins (in order of binding) Origin Recognition Complex (ORC) multisubunit protein complex bind to replication
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Eukaryotic chromosomes are linear not circular - multiple origins of replication, not just one # DNA must be unwound # nucleosomes disaggregate and separate from DNA Eukaryotic DNA Replication Starts at the origin of replication # up to 60 origins /chromosome # 1 origin / 300kilobases Mechanism of initiation # Initiator proteins (in order of binding) Origin Recognition Complex (ORC) multisubunit protein complex bind to replication
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October 7, 2009 Lecture 4 – DNA Synthesis and Replication
DNA Synthesis happens during S (synthesis) Phase of the Cell Cycle
○ Semiconservative replication ■ 2 x (1 parent(old) strand + 1 daughter(new) strand) ○ Eukaryotic VS Bacterial DNA replication ■ Eukaryotic chromosomes are linear not circular ● → multiple origins of replication, not just one ■ Slower, 50-75 nucleotides/sec, would take 800 h ● → multiple origins of replication, not just one ■ DNA must be unwound ■ Nucleosomes disaggregate and separate from DNA
● Eukaryotic DNA Replication
○ Starts at the origin of replication ■ up to 60 origins/chromosome ■ 1 origin/ 300kilobases ○ Mechanism of initiation ■ Initiator proteins (in order of binding) ● Origin Recognition Complex (ORC) multisubunit protein complex bind to replication origin ● Minichromosome maintenance (MCM) proteins include DNA helicases which unwind DNA ○ Helicase loaders help MCM's bind to ORC ● = Prereplication complex ● DNA now “licensed” for replication ○ Replication bubbles ■ Bubbles meet and fuse to make the new DNA ● 2 new strand of DNA, nucleosomes rapidly reform, new DNA protected from degraedation ● in the middle of the bubble is the origin of replication
● Phosphdiester Bond Synthesis
○ dATP, dGTP, dCTP, TTP are substrates in DNA synthesis ■ added to growing 3' tail of copy ■ binds by A:T & G:C rules ■ Bond formation is a nucelophilic attack ● attacking group is the 3' OH of deoxyribose dATP ● nucleophilic attack - a chemical compound that is attracted to nuclei and tends to donate or share electrons ● ά phosphoryl of the incoming dNTP is attached ● β and γ phosphoryl groups are liberated as PPi (drives polymerisation) ● Replication Fork ○ Helicase ■ unwinding DNA ○ RPA ■ ss binding protein ○ Primase ■ synthesizes RNA primer ○ DNA Polymerase α ■ loads bases on to lagging template strand ● Synthesis of the leading DNA Strand 1. Helicase unwinds and opens ds-DNA at the replication fork 2. RPA 's coat the leading and lagging parent strands 3. RNA Primase and DNA polymerase α load onto template strand 4. Primase makes RNA primer (~10bases) 5. DNA polymerase α replaces primase 6. DNA tract (~10-30bases) is synthesized 7. DNA polymerase α stops synthesizing 8. DNA polymerase α is replaced by DNA polymerase δ ■ AKA Polymerase Switching - initiated by replication factor C (RFC) 1. RFC powered by ATP hydrolysis makes DNA polymerase α dissociate from template strand 2. RFC makes proliferating cell nuclear antigen (PCNA) bind to the end of the DNA tract by opening the toroid and encircling the DNA double helix ○ PCNA - a toroidal (doughnut-shaped) homotrimer (made of 3 identical monomers) of 36-kDa subunits ■ works as a sliding clamp holding the polymerase to the primer terminus 3. PCNA makes DNA polymerase δ bind to the template strand 9. DNA polymerase δ synthesizes thousands of nucleotides with out dissociating ■ high processivity ● Synthesis of the lagging DNA Strand ○ essentially the same as synthesis of leading strand ○ BUT, polymerases synthesize DNA away from the replication fork ○ → DNA is synthesized in many small segments (Okazaki fragments) away from the replication form ○ DNA ligase joins the Okazaki fragments
● Removal of RNA primers
○ Rnase H Model ■ Rnase H1 cleaves the primer RNA one nucleotide upstream of the RNA-DNA junction ■ RNA is removed intact leave 1 ribonucleotide on the downstream DNA portion of the Okazaki fragment ■ FEN1/RTH1 removes the remaining junctional nucelotide ■ DNA strand is sealed by DNA ligase I ○ Flap Model ■ FEN1/RTH1 binds at 5'end of the RNA tail and slides down DNA seperating the RNA primer from the DNA → a flap forms ■ FEN1 cuts the at the branch point (RNA primer- new DNA) ■ DNA Polymerase δ fills where the RNA primer used to be ■ The gap between the 2 new DNA fragments is sealed by DNA ligase ● Problems encountered during replication (solved by reactions at the replication fork) ○ Continued opening of a replication bubble would eventually cause knotting of the dublex DNA, how is double stranded DNA unwound? ■ DNA Helicase ● its function is to unwind DNA ● parts the helices ● binding tightening when dTTP binds to helicase ● helicase rotates as the dTTP I hydrolysed ● helicase is stimulated by ssDNA-binding proteins called replication protein A (RPA) ○ DNA polymerases cannot initiate strand synthesis, how is DNA synthesis initiated? ■ RNA primase synthesizes a short RNA tract(primer) of about 10b- 30b ■ replication bubble forms at the origin of replication ■ Primase binds to the initiation point on the leading and lagging parent chain ○ DNA polymerases synthesis only in the 5' → 3' direction, how is the lagging strand copied? ■ The lagging strand is looped ■ ○ DNA must be copied with high fidelity, how is DNA synthesis accuracy maintained? ■ DNA polymerase δ posses exonucleoase activity ● 5'-3' exonuclease ○ removes DNA strands that are in the way of DNA polymerase ● 3'-5' exonuclease ○ proof-reads activity and removes mis-incorporated bases
● Regulation of DNA Replication
○ Positive regulation of replication ■ LICENSING ● ORC stays on DNA throughout replication ● Licensing factors Cdc6 + Cdt1 accumulate during G1 and bind to ORC ○ essential for coating DNA with MCM ● Only DNA with MCM is replicated ● Cdt1 + Cdc6 leave the ORC once replication starts ● MCM leave in front of the replication fork ○ Negative regulation of replication ■ Nuceli in phase G2 synthesize geminin ● Geminin may separate Cdt1 ○ MCM proteins can't assemble ○ DNA can try again at the next S phase (Geminin is degraded at the end of mitosis)