Biochemistry
Chen Yonggang
Zhejiang University
Schools of Medicine
Central dogma
replication
transcription translation
DNA
RNA
Protein
DNA Replication
.
DNA Replication-Conservation of
Information
• DNA replication must be carried out every
time a cell divides
• Procaryotic growth involves cell division
• Mitosis in eucaryotes involves cell division
• DNA replication is template driven and
synthesizes DNA in a semi-conservative
manner
dNTP + DNAn
DNAn+1 +PPi
DNA is replicated in a semiconservative manner
• Messelson & Stahl showed, using 15Nlabelled DNA that the products of
replication had intermediate density McKee 18.2
strand A
strand B
15N 15N
14N
15N 14N
14N 15N
14N
15N14N 14N 14N
14N
15N14N
14N 14N
Each separated DNA strand
is duplicated to give two
new double helices.
DNA semiconservative
replication
Each DNA strand serves as a template for
the synthesis of a new strand, producing
two new DNA molecules , each with one
new strand and one old strand, this is
semiconservative replication.
The process which appeared
simple initially is complex
• Replication occurs just prior to cell division
• The E coli chromosome is a single circular
DNA double helix associated with proteins
in a nucleoid
• The E coli chromosome is negatively
supercoiled and thus is quite compact and
inaccessible
McKee 17.16
To allow the replication to occur
supercoiling must be relaxed
• A type I (single strand breaking) topoisomerase
cleaves and relaxes the negative supercoiling
ahead of the replication complex
• The topoisomerase has a central hole through
which the double helix passes. An intermediate is
an enzyme-linked 3’OH
• dnaA is displaced providing access for the next
component needed for replication
The first step in replication
involves oriC
• OriC is a 245 bp region, the origin of E coli
replication
• OriC contains 3 tandem repeats of a 13 bp
sequence beginning in GATC and rich in AT bp
• These repeats are weakly H-bonded and serve to
provide 4 binding sites for a protein, dnaA, a start
signal for replication
• Replication proceeds in two directions bidirectional
dnaA allows binding of two other
important proteins
• dnaB is a DNA helicase which carries out
the ATP- driven unwinding of the DNA
double helix
• dnaC is an important accessory protein
which binds and is soon released
• Together the three proteins utilize ATP to
bend and separate the two strands of the
bacterial chromosome
SSB, a single strand binding tetramer
stabilizes the initiation complex
McKee 18.7
DNA replication involves many
enzymes
• An RNA primase binds to the SSB
stabilized melted helix
• Since DNA polymerases require a primer
and only extend that primer, the RNA
primase (dnaG) in association with other
proteins (primosome) synthesizes a 5-7
nucleotide primer using information from
the template strand
At the replication fork two
strands are managed differently
• The 5’ end of the primer(leading strand) is
extended continuously by DNA polymerase
III in a 5’→3’ direction by dNTPs
• The primer on the lagging strand is also
extended 5’→3’ by DNA polymerase III, in
a discontinuous manner
• Thus primer is made once on the leading
strand and every 1000 nucleotides on the
lagging strand
The replication fork
McKee 18.6
DNA Polymerase III holoenzyme
contains 10 distinct types of subunits
• DNA Polymerase III is the primary replicase in E.
coli
• It has polymerase and 3’→5’ exonuclease
activities
• It functions as a dimer
• It has great fidelity, only 1 error in 1010 bp
• It is highly processive, sticking to the DNA for the
entire trip through the chromosome
• It has a rapid biosynthetic rate, synthesizing 1000
nt/sec
The Pol III synthesizes DNA
from dNTPs
McKee 18.3
The Dimer moves in one
direction and synthesizes 5’→3’
• The leading strand is synthesized by
addition of 5’-dNTPs in response to the
template
• The looped lagging strand is synthesized in
Okazaki fragments using 5’-dNTPs
• The lagging strand must be pieced together
using DNA Polymerase I
DNA polymerase III forms
phosphodiester bonds
• 2’-deoxynucleoside 5’ triphosphates are
the activated intermediates needed for
synthesis
• Information from the parental strand
provides the information for 5’→3’
synthesis (parental strand is read 3’→5’)
• Thus each parental strand serves as the
template for synthesis of a complementary
strand
Synthesis of a phosphodiester
bond
O
CHAIN P O
O
BASE1
O
nucleophilic attack
O
O
O
O
CHAIN P O
O
O
.. OH
O
O
O P O P O P O
O
O
O
OH
BASE2
O P O
BASE1
new
phosphodiester
bond
BASE2
O
O
OH
DNA Polymerase III is at the
center of Replication
McKee 18.8
Top-down view of replication
While DNA polymerase III does
the replicating, DNAP I cleans up
• Pol I(100kd) is a monomer of about 10%
the size of Pol III(900kd)
• It has three activities
– It is a DNA polymerase
– It is a 3’→5’ exonuclease
– It is a 5’→3’ exonuclease
• It is a processing and proofreading enzyme
The three activities are on one
polypeptide
• The larger fragment(Klenow fragment) of
67kd contains the polymerase and the
3’→5’ exonuclease activity
• The smaller, 36kd contains the 5’→3’
exonuclease activity
As Pol III finishes, Pol I goes to
work
• The 5’→3’ exonuclease removes the RNA primer
• The polymerase synthesizes DNA to fill the gap
• Errors in Pol III synthesis are removed by 3’→5’
exonuclease
• The function of DNA Pol II is not understood,
although it apperas to be similar to Pol I
Supercoiling was taken out by
dnaB, DNA gyrase replaces it
• Following synthesis of the strands, excision
of RNA, replacement by DNA using Pol I,
the supercoiling can be reinstated
• DNA gyrase, an ATP- linked, energy
requiring enzyme introduces negative
supercoils to restore the original twist in the
leading strand
DNA ligase seals the Okazaki
fragments and the completed double
helical DNA
• DNA polyI removes the primers and fills the gaps,
DNA ligase seals the nicks and Okazaki fragments
are connected
• Pyrophosphate cleavage drives the reaction to
completion
• Termination occurs at a ter region and is mediated
by a binding protein TBP
• A type II(double stranded) topoisomerase is
probably involved in helix dissociation(the two
daughter DNA molecules separate)
Enzymes and proteins involved in DNA
replication
1, Topoisomerase
2, dnaA 1 : recognize the origin of replication
dnaB(helixase):unwind double helix
dnaC
3, SSB
4, Primase
5, DNA polymeraseIII, DNA polymeraseI
6, Ligase
Happy Birthday
•Samar and Barry
Eucaryotic Replication is similar
to that of procaryotes
• Both have initiation, elongation and termination
phases and are bidirectional
• Both involve multiple DNA polymerases
• Both involve multiple copies of the primary
replicase which replicates strands differently
• Replication rate is slower, but replication is rapid
due to multiple replicons
• Both require topoisomerases to unwind and
rewind the DNA
• Both require ligases
Eucaryotic replication is distinct
from that of procaryotes
•
•
•
•
There are 5 polymerases(α,β,γ,δ,ε)
The chromosomes are linear
There are multiple ori, and replication units
Replication only occurs during the S phase
of the cell cycle
• Telomeres restrict the number of times a
replicon can be expressed
Initiation of replication occurs at
multiple ori
• A large complex of proteins assembles at an ori
(Origin Recognition Complex-ORC)
• Details not for testing
– A complex with helicase activity must bind and be
activated
– Replication Protein A (RPA ) binds and separates the
strands(like SSB in E.coli)
– RFC(replication factor C– a clamp loading factor) and
PCNA(proliferating cell nuclear antigen) allows
binding of Pol d to both the leading and lagging strand
Binding of initiation factors to
the lagging strand differs
• Pol d is the main eucaryotic replication
polymerase (Details not for testing)
– Replication protein A(RPA) binds to the single strands
– Pol a and a primase complex binds to the lagging strand
– An RNA primer and 15-30 dNTPs are synthesized
• Pol d binds and replicates one nucleosomes worth
of Okazaki fragment
Finishing and sealing of the
lagging strand is different
• Specific protein factors are important in finishing
up replication (details not for testing)
– DNA polymerase  remove the primers and DNA
polymerase d excise errors
– Topoisomerases induce supercoiling
– DNA ligase seals the breaks
– Chromosomes segregate
• Replication bubbles merge
• Telomeres determine the end of replication
Eukaryotic DNA polymerases
• Eukaryotes have at least 15 DNA Polymerases (5
most important):
Pol α: acts as a primase (synthesizing a RNA primer),
and then as a DNA Pol elongating that primer with
DNA nucleotides. After a few hundred nucleotides
elongation is taken over by Pol δ and ε.
Pol β: is implicated in repairing DNA.
Pol γ: replicates mitochondrial DNA.
Pol δ: is the main polymerase in eukaryotes, it is
highly processive and has 3'→5' exonuclease
activity.
Pol ε: may substitute for Pol δ in lagging strand
synthesis, however the exact role is uncertain.
Telomeres are GC rich selfcomplementary sequences at
chromosome ends
• Telomerase maintains the telomeres
• Telomeres are repeat structures with a
terminal loop
• At each replication the telomeres are
modified using an integral RNA template
• Loss of telomeres limits replication
• Cancer cells lose control of their telomeres
Telomeres
The structure at the ends of linear eukaryotic
chromosomes, generally consist of many
tandem copies of a short oligonucleotide
sequence.
Supercoiled DNA in Prokaryote
Structure of Nucleosome
Negative and positive supercoil