Lecture 3 Flashcards
DNA polymerase error rate in vitro
1/10^4 - 10^5
Tautomeric base conformation during addition
Proofreading mechanism
Same polymerase has different active site
Exonuclease that unravels 3-5 direction
With base selection : accuracy is 1: 10^6-8
Most common dna polymerase in E Coli
Rate of addition
DNA polymerase I (5 total types)
600 bp/min in vitro
But true measurement is 60,000 bp/min
Klenow fragment
Protein fragment of DNA polymerase I
Mild protease treatment = exonuclease segment removed which removes primers
DNA polymerase I subunits
9 total
Alpha = polymerization
E = proofreading
3x Alpha + e linked together by clamp loading complex
DNA polymerase III subunits
16 total
Much greater processivity than dna Pol I
Helicases
Strand separation
Use atp
Primase
RNA primer synthesizer
Dna ligase
Seals nick
Uses atp
Functions of Pol I
5-3 exonuclease = nick translation, rna primer removal
3-5 exonuclease - proof reads step by step during addition - induced kinetic pause if mismatch
5-3 polymerase
Pol I processivity and rate
20-100 nt
20nt/second
Pol III processivity and rate
3-5 exonuclease activity
> 500,000 nucleotides
250-1000nt/s
Sequences in Ori C
DUE = at rich
R binding sites
I sites = dna must be complexed w atp to bind
DnaA protein
Binds to ori C and starts strand separation
DnaB
Primary helicase during replication
5-3
What is the only regulated phase of the cell cycle?
Initiation
Dam methylase
Methylates n 6 position of adenine
Right after replication, new strand yet to be methylated
All neeeds to be methylated for new round of initiation = DnaA binding
Length of rna primer
10-60bps
What is DnaB physically associated with during replication
Lagging strand dna POL III
Leading strand dna POL III
Clamp loading complex
Sometimes primase
DnaA analog
ORC
DnaB analog
MCM2-7
Additional initiation reagents
Cdc6
Ctd1
Cdc45
GINS
DNA polymerase e
Analog to DNA polymerase III
DNA polymerase a
Analog to primase / pol I
Adds rna primers and extends them
No proofreading
PCNA
Proliferating cell nuclear agent
Large amounts in proliferating cells
Analog of beta subunit Clamp
Clamp loader analog
RFC
SSB Analog
RPA
Lagging strand in eukaryotes
Polymerase delta
Acyclovir
Compound to inhibit herpes viral polymerase
Incomplete ribose ring
No 3’ OH for nucleophilic attack
Dogma for information transfer
DNA to rna to protein
Also
RNA to dna
Reverse transcriptase
RNA dependent dna polymerase
Uses rna as template
Degrades rna part of double strand product
Replaces with dna
Each have different active site
No proof reading
Retro viruses
Retroviruses have 3 genes
Gag
Pol
End
Ends have LTR to facilitate integration
tRNA primer
Needs to already be on dna from earlier infection
Transposons
Transpose positions via rna intermediate
