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Human Molecular Biology

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)

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