15: DNA replication Flashcards
replication is
semi conservative
steps of replication?
- initiation
- priming
- DNA synthesis (+ proofreading)
- ligation
What are the characteristics of bacterial initiation?
1 replication origin that is highly conserved, thus easy to find, and tends to be A=T rich bc easier to pull apart
single stranded binding proteins prevent DNA from becoming double stranded again
replication bubble = 2 replication forks
other initiation proteins do initial separation of ds then helicase can bind
helicase: 6 subunits, circular with hole in middle that is large enough for single strand; unzips DNA strands by dissociated NCPs using ATP energy causing strands to be physically separated
What are the characteristics of eukaryotic initiation?
multiple replication origins (more DNA)
SSBP, helicase
What are the characteristics of priming?
primase binds to helicase and synthesizes a short (10-20nt) strand of RNA
primase is not perfect bc thermodynamically hard to fit right base – makes mistakes
3’ end grows (currently hybrid RNA/DNA structure)
sliding clamp binds to RNA/DNA hybrid and is large enough for double strand section
DNA polymerase binds to clamp (DNA poly can bind for longer with clamp) – structurally similar in bacteria and humans
What are the characteristics of DNA polymerase III?
DNA polymerase III extends primer from 3’ end only in bacteria
DNA polymerase III pushes SSBP away
DNA poly III has active site for DNA synthesis: nucleoside triphosphate added to strand (hand structure)
-> structural constraints that only allows correct base to make phosphodiester bond, poly tries out diff bases at random until correct rxn occurs
How does DNA synthesis occur chemically?
O of OH on 3’ C of sugar nucleophilic attack on phosphate of dNTP
spontaneously cleaves pyrophosphate an forms phosphodiester bond
What does acyclovir (antiherpes) do to target viral DNA synthesis?
antiviral drug - similar to nucleoside structure - contains guanine base = gets inserted to DNA replicates but no free 3’ OH (and not cyclic) = no nuc attack = replication terminated
What does zidovudine (AZT, anti-HIV) do to target viral DNA synthesis?
antiviral drug - similar to deoxythymine phosphate but it has an azide group (N3) instead of 3’ OH
get phosphorylated, this will get added to nucleotide chain and stop replication (no nuc attack)
What does the bacterial replication fork look like?
leading strand (template 3’-5’ and synthesize 5’-3’)
lagging strand (template 5’-3’ and synthesize 3’-5’) - trombone loop
repliosome: protein complex, when DNA poly dissociates, no loop
at least 2 polymerase associates with each helicase (1 in leading and 2 in lagging, Okazaki fragments can replicate at the same time)
What is the DNA polymerase proofreading characteristics and mechanism?
improves error rate to 1 in 10^6 or 10^7
- disincorporated nucleotide bc OH is not is right position to add next nucleotide
- 3’->5’ exonuclease enzyme degrades nucleic acid backbone
- DNA polymerase stalls and adds correct base
How is the RNA primer removed in bacteria?
- DNA poly 3 dissociates and DNA poly 1 binds
- DNA poly 1 has 3’-5’ activity (exonuclease activity to fix own mistakes) and 5’-3’ activity (degrades RNA in front)
- DNA poly 1 synthesizes replacement DNA (causing nick translation - nick was infant of RNA on 5’ end, and is now after newly synthesized replacement DNA)
How is the RNA primer removed in eukaryotes?
- Rnase H degrades primer while regular replicative DNA polymerase fills gap, nick translation
What are the types of DNA polymerase? Where are they found? What are their functions?
DNA poly 1: Bacteria, replaces primers
DNA poly 3: Bacteria, DNA synthesis
replicative DNA poly: eukaryotes: replaces primers and DNA synthesis
What is the ligation mechanism? How is it different for eukaryotes and prokaryotes?
- DNA ligase joins strands at nick (backbone break) by binding to sliding clamp
- energy requiring: eukaryotes and some bacteria hydrolyze ATP; most bacteria split NAD+
