DNA replication Flashcards
Which types of proteins are involved in DNA synthesis and what is their function?
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What are the main differences btw pro- and eukaryotic DNA replication?
in eukaryotes:
- many ORC (instead of oriC) b/c much lower rate of replication → replication bubbles instead of replication fork
- SSBs are called replication protein A (RPA)
- different DNA polymerases
- DNA has to be rearranged into chromosomes after replication
Compare speed and error rate (fidelity) of DNA replication (in pro-/eukaryotes), RNA transcription and protein translation.
- DNA replication: very low error rate (high fidelity) ~ 10-8 - 10-9, a bit slower in eukaryotes due to nucleosomal interactions
- RNA transcription/translation: slower than replication, much higher error rate ~ 10-4
Differentiate btw prokaryotic DNA polymerases.
Which ones are involved in DNA replication?
- Pol I: DNA replication (RNA primer removal), DNA repair
- Pol II: DNA repair
- Pol III: DNA replication
Which factors contribute to the high fidelity of DNA replication in prokaryotes?
-
intrinsic fidelity of DNA polymerase: 10-3 - -4
sensing dNTP complementarity to template -
exonuclease proofreading of DNA polymerase: 10-2 - -3
sensing primer complementarity to template -
mismatch repair: 10-2
sensing complementarity of 2 strands, distinguishing parental/daughter strands
⇒ overall: 10-8 - 10-9
Differentiate btw eukaryotic DNA polymerases.
Which ones are involved in DNA replication?
- Pol α: DNA replication (primer synthesis) = primase
- Pol β: base excision repair
- Pol γ: mitochondrial DNA replication/repair
- Pol δ: DNA replication (lagging strand synthesis), nucleotide/base excision repair
- Pol ε: DNA replication (leading strand synthesis), nucleotide/base excision repair
Which reaction is catalyzed by DNA polymerases (in eukaryotes and prokaryotes)?
dNTP + OH- on 3’ end as primer
→ DNA polymer w/ phosphodiester bonds + PPi
BUT: require template strand + Mg2+ as cofactor
⇒ synthesizes 3’→5’, daughter strand formed 5’→3’
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How are both DNA strands synthesized?
- leading strand synthesized continuously
- lagging strand synthesized discontinously, Okazaki fragments are formed (in prokaryotes ~ 1000 bps, in eukaryotes ~ 200 bps)
⇒ bidirectional synthesis in replication bubble
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How is mismatch basepairing recognized by DNA polymerases?
DNA polymerase recognizes differences in atom distances + bond angles
What is special abt DNA pol I?
- coded by pol A: mutants lack proofreading activity
- monomer
only prokaryotic DNA pol w/ 3 activities b/c involved in replication and repair
- 3’→5’ polymerase activity
- 5’→3’ exonuclease (essential for initial removal of RNA primer)
- 3’→5’ exonuclease (proofreading)
How does DNA pol I know when to use its polymerase or proofreading activity?
Klenow segment of DNA pol I has P and E site
- when template connected to primer passing through P site: polymerisation
- when template unwinds from primer + translocates to pass through E site: editing
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What is the function of DNA pol II?
participates in repair, requires duplex template and primer
- coded by pol B: mutation is not lethal
- multimeric (4+)
only 3’→5’ exonuclease activity (proofreading)
Describe the function of the structural elements of DNA pol III.
2 DNA pol III form an asymmetric dimer w/ multiple subunits
- each α subunit: 3’→5’ polymerase activity
- β subunits: act as sliding clamp, keep enzyme attached to DNA
- γ subunit: acts as clamp loader for lagging strand Okazaki fragment, help β subunits to form unit + bind to DNA
- each ε subunit: 3’→5’ exonuclease activity (proofreading stimulated by Θ subunit)
- τ subunits: dimerize, tether both enzymes together
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What is the function of DNA pol III?
replicates prokaryotic DNA
- coded by pol C: mutants are temperature sensitive, conditionally lethal
3’→5’ polymerase activity +
3’→5’ exonuclease activity (proofreading)
What is the mechanism of 5’→3’ exonuclease activity of DNA pol I?
removes RNA primer on lagging strand + fills in the necessary nucleotides between the Okazaki fragments in 5’→3’ direction, proofreading for mistakes as it goes