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
