DNA REPLICATION
Types of DNA replication:
A.
Prokaryotes
B.
Eukaryotes
Phases of DNA replication: these 3 phases occur for
all polymerization processes:
1.
Initiation
2.
Elongation
3.
Termination
DNA replication proteins (the main players in this
process)
-
Topoisomerase/DNA Gyrase?
-
Helicase
-
Single Stranded Binding Proteins (SSBP or just
SSB)
-
Sliding/DNA Clamp and the Clamp loader
a.
Prokaryotes: β-clamp
b.
Eukaryotes: Proliferating cell nuclear antigen (PCNA) –
sliding clamp for eukaryotes and archaea and Replication factor C (RFC) is the
clamp loader
-
Primase
-
DNA POLYMERASE (replicative DNA polymerases:
a.
bacteria: III
b.
eukaryotes:
δ leading strand, ε lagging, α replacing RNA primer with DNA?, γ mitochondrial replication)
-
DNA Ligase
-
Telomerase (eukaryotes)
-
Flap endonuclease (FEN-1) (eukaryotes)
-
Other players:
a. Prokaryotes
-
Prokaryotic proteins/factors and subunits for
polymerase III holoenzyme???: DnaA, DnaB, DnaC, DnaE, DnaG, DnaH, DnaI, DnaN,
DnaQ, DnaS, DnaX
b. Eukaryotes
-
Histone acetylase - +ve charge on lysine
residues relaxes superstructure/deacetylase??? .
-
Mini-chromosome maintenance (MCM): 6 MCM
subunits MCM 2-7 that form a complex (eukaryotes)
-
Ribonuclease: RNase H
-
Replication protein A (RPA)
-
Origin Recognition Complex (ORC) (eukaryotes)
Random terms, concepts and players:
-
Proofreading
-
Processivity
-
Okazaki Fragments
-
High-fidelity
-
Replication bubbles/eyes
-
RNA primer
-
Leading strand and lagging strand (remember
though that left and right of the origin, replication is )
-
Origins: ori: oriC in E.Coli of AT 13
nucleotides and in eukar
-
Replication fork
-
Pre-replication complex (in pro- and eu-):
-
Licensing factor
-
Telomere: protect chromosome ends 6-mer repeats of
TTAGGG nucleotides
-
Primosome
-
Klenow fragment
-
Replisome (bact-)
-
Chromatin during replication (eu-): half of H3,
H4 go to one daughter strand and half to the other: parental histone
segregation ASF1. Chromatin assembly
factor 1 (CAF-1) deliver new histones to the replication fork
-
Trombone model of the lagging strand
-
Termination: when replication forks meet in
circular dna, Ter site that is bound by Tus protein. In eukaryotes it goes to
the end except from the end problem of lagging strand
Features:
-
Semiconservative: not conservative nor
dispersive
a.
Messelson and Stahl experiment: Solution with
N-15, leave to multiply E.Coli, put a small sample into N-14. Take a small
sample of extract DNA add CsCl 1.7g/cm3 then centrifuge and show from the
pattern that is conservative. Important elegant experiment in history need that
need to know
-
Bidirectional: J. Cairns in 1963 autoradioraphy
and θ structure
in bacteria
-
Semi-discontinuous: continuous on leading –
although due to repairs there are some ‘’gaps’’, discontinuous on lagging
-
One origin in prokaryotes, multiple origins in
eukaryotes (thousands firing at different times)
-
Pulse-chase experiment?? For RNA transcription???
-
C-G three hydrogen bonds and A-T two;
complementary strands
Replication of organelle DNA
-
Mitochondrial
Replication of plasmids/bacteriophages/viroid: Rolling circle replication:
unidirectional
Regulation of DNA rep.:
-
Prokaryotes: RIDA regulation inactive DnaA,
SeqA,
-
Eukaryotes: cyclin?
DNA polymerases:
-
7 families: A, B, C, D, X, Y, RT (reverse
transcriptase)
-
bacteria: 5 DNA polymerases; I II III IV V
-
eukaryotes: 14 DNA polymerases: α,
β, γ, δ, ε, ζ, η, θ, ι, κ, λ, μ, ν, Rev1
DNA polymerase III: HolA, HolB, HolC, HolD,
HolE
All polymerases can extend strand at the 3’ end because they
require a OH to add the nucleotide so for this reason primase adds a short
stretch of RNA (primer) to allow polymerase to take it from there.
-
5’->3’ synthesis but read in 3’->5’ way
