Download The Replication of DNA

The Replication of DNA
School of Life Science
Shandong University
Ch 8: The replication of DNA
1. Semiconservative replication
2. The chemistry of DNA synthesis
3. The mechanism of DNA polymerase
4. The replication fork
5. The specialization of DNA polymerases
6. DNA synthesis at the replication fork
7. Initiation of DNA replication
8. Finishing Replication
9. The regulation of genome replication
1. Semiconservative replication
When
Watson and Crick proposed the double
stranded helix for the structure of DNA, they
suggested that it might replicate by having the
two parental strands separate and each one
acting as a template for a new strand---semiconservative replication.
There are 3 possible mechanisms of DNA
replication.
Page 27-28
Replication is semi-conservative
2. The chemistry of DNA synthesis
2.1 DNA synthesis requires
deoxynucleoside triphosphates
and a primer:template junction
dNTP
p196 Fig.8-1
A primer is a short piece
of RNA that provides a 3’OH for DNA synthesis.
2.2 DNA is synthesized by extending the 3’ end of
the primer. Hydrolysis of pyrophosphate (PPi) is the
driving force for DNA synthesis
p197 Fig. 8-2
5’
3’
3. The mechanism of DNA polymerase
DNA polymerase resemble a
hand that grips the
primer:template junction
3.1 DNA Polymerase use a single active site
to catalyze DNA synthesis.
Recognition of different dNTP
by monitoring the ability of
incoming dNTP in forming A-T
and G-C base pairs (nearly
identical geometry); incorrect
base pair dramatically lowers
the rate of catalysis (kinetic
selectivity.
p199 Fig. 8-3
Distinguishing between rNTP and dNTP by steric exclusion
of rNTPs from the active site.
p199 Fig. 8-4
分辨器氨基酸
细胞中rNTP的浓度
比dNTP的高约10倍，
但前者的掺入速率
却是dNTP的千分之
一。
——通过动力学选择效应及空间排
阻效应以保证参入碱基的正确性
3.2 The palm domain of DNA Polymerase:
Catalytic domain
Page204
The palm domain binds two divalent metal ions (typically Mg+2 or
Zn+2) that alter the chemical environment around the correctly
base-paired dNTP and the 3’-OH of the primer
3.3 DNA polymerases are processive enzymes
Each DNA polymerase
has a characteristic
processivity that can
range from only a few
nucleotides to more than
50,000 bases added per
binding event.
Page206
3.4 Exonuclease proofread newly synthesized DNA
Proofreading exonucleases
work like a “delete key”
exonuclease: degrade from a end
endonuclease: cut in the middle
Page208
4. The replication fork
The junction between the
newly separated
template strands and
the unreplicated DNA
duplex is called the
replication fork.
4.1 Both strands of DNA are synthesized
together at the replication fork.
Leading strand
3’
5’
5’
3’
Page209
Lagging strand
Fig. 8-11
The short fragments of new DNA in lagging strand are called Okazaki fragments
and vary in length from 1000 to 2000 nucleotides in prokaryotes and from 100 to
400 nucleotides in eukaryotes .
Semidiscontinuous replication
（半不连续复制）
——Okazaki fragments
Okazaki did his experiments in bacteriophage
T4, which infects E. coli.
He added [3H] thymidine to the infected E. coli
cultures. Then the DNA was isolated and
treated with a strong base to make it single
stranded. It was separated by size using CsCl
centrifugation. This allowed Okazaki to
separate small pieces of DNA from larger ones.
4.2 The initiation of a new strands DNA requires an
RNA primer
The first few nucleotides at the 5’-end of Okazaki
fragments are ribonucleotides. That is then removed
before fragments are joined. Crucial for high fidelity
of replication
Primase (引发酶) is a specialized RNA polymerase
making short RNA(5-10) primers that is
complementary to one ssDNA.
Remember that DNA polymerase requires a 3’ OH
to begin DNA synthesis.
 The leading strand will need one primer and then the
rest of the DNA template can be copied by DNA
polymerase.
 The lagging strand will require a primer for each
Okazaki fragment.
4.3 RNA primers must be removed to complete
DNA replication——RNase H
RNase H is a specific RNase that
removes RNA from RNA:DNA hybrids. It
leaves a the last RNA base pair (can
only cleave bonds between two
ribonucleotides), which must be
removed by a 5’ exonuclease. (This 5’
exonuclease is part of DNA pol I in
E. coli.)
DNA pol I
Figure 8-12
4.4 DNA helicases unwind the double helix in
advance of the replication fork
The DNA strands are separated by DNA helicase (解旋酶).
It forms a ring around a single strand of DNA and slides
along the DNA using energy from the hydrolysis of ATP. DNA
helicase is also called DnaB (E. coli).
Page 212
4.5 Single-stranded binding proteins (SSBs)
stabilize ssDNA prior to replication
SSBs bind to the single-stranded DNA to stabilize it. SSBs
prevent the hydrogen bonds from reforming.
The binding of a single SSB to the single-stranded DNA helps the
binding of another SSB. This is called cooperative(协同作用)
binding. Which helps the SSBs to quickly cover and stabilize all
the single-stranded DNA.
The SSBs do not bind to a specific sequence, but have sequence
independent binding.—— Sequence-independent manner
Figure 8-15
4.6 Topoisomerase removes supercoils produced
by DNA unwinding at the replication fork
As the strands of DNA are separated at the replication fork,
the dsDNA in front of the fork develops positive supercoils.
（复制机器）
Fig. 8-16
As DNA replication occurs positive supercoiling occurs.
A type II topoisomerase (also called DNA gyrase) makes
2 cuts in the DNA to cause negative supercoiling to
compensate for the positive supercoiling caused by
replication.
Fig. 8-16
Topoisomerase II
5. The specialization of DNA polymerases
5.1 DNA polymerases are specialized for different
roles in the cells
There are different DNA polymerases that are specialized
for different situations. Some are important for
chromosome replication and others are important for
DNA repair.
E. coli has 5 different kinds of DNA polymerases. They
are named with Roman numbers in the order they
were discovered.
Each of the polymerases is specialized for a different
role in the cell.
Eukaryotic cells have multiple DNA polymerases.
Three are essential to duplicate the genome: DNA Pol
δ, DNA Pol ε and DNA Pol α / primase (引发酶)
 DNA polymerase III (DNA Pol III) is the main enzyme that
replicates the chromosomes. It is highly processive (once it
binds to the DNA, it adds many base pairs before it
dissociates).
 DNA polymerase I (DNA pol I) is specialized for removing the
RNA primers. It has a 5’ exonuclease that removes RNA or
DNA directly in front of newly synthesized DNA.
The 5’ exonuclease can remove the RNA-DNA linkage that is
resistant to RNase H. (We will discuss the RNase H later.)
 DNA pol I can also add nucleotides to the DNA strand. But it
is not highly processive. It adds 20-100 nucleotides after
binding. The short extent of synthesis is suited for replacing
the region previously occupied by the RNA primers.
 Both DNA pol I and DNA pol III are important for DNA
replication. They must be very accurate, so both
have a proofreading exonuclease.
 The other 3 DNA polymerases in E. coli are important
for DNA repair and lack proofreading capabilities.
DNA Pol α / primase (引发酶)
primase: synthesize an
RNA primer
then replaced by DNA Pol δ
(lagging strand),
and DNA Pol ε (leading
strand)
Fig. 8-17 DNA
polymerase switching
during Eukaryotic DNA
replication
5.2 Sliding clamps (滑动加环/滑动钳) dramatically
increase DNA polymerase processivity
Clamp protein slides
along the DNA without
dissociating from it.
Sliding DNA also bind tightly to DNA
polymerases at replication forks.
After DNA polymerase has
completed synthesis of the
template, the absence of a
primer:template junction
causes a change in the DNA
polymerase that releases it
from the sliding clamp.
5.3 Sliding clamps are opened and placed on DNA
by clamp loaders
ATP control of sliding
DNA clamp loader
6. DNA synthesis at the replication fork
In E. coli,
DNA Pol III holoenzyme（全酶）
Enhance the function of core
enzyme
Includes two copies of the “core”
DNA Pol III enzyme and one copy of
the five-protein γ complex
Page 227
Figure 8-22
Protein interaction
e.g.
a) helicase—Pol III
holoenzyme
b) helicase—primase
E. coli trombone model
Topoisomerase II
The combination of all the
proteins that function at the
replication fork is referred to as
the replisome（复制体）, a
finely tuned factory for DNA
synthesis with the activity of
each protein is highly
coordinated.
7. Initiation of DNA replication
7.1 Specific genomic DNA sequences direct the
initiation of DNA replication
The initiation of replication requires the separation of the
two strands of the DNA duplex to provide ssDNA.
DNA synthesis generally initiates at internal regions.
The specific sites at which DNA unwinding and initiation of
replication occur are called origins of replication.
All the DNA replicated from a particular origin of
replication is defined as a replicon.
7.2 The replicon model of replication initiation
 Often there are other sequences
besides the origin of replication that
are important for initiating replication.
 All the DNA sequences that are
sufficient to direct the initiation of DNA
replication are given the name
replicator.
 The initiator is a protein that
specifically recognizes the DNA
sequences in a replicator and initiates
DNA synthesis.
Please read p230
Fig. 8-24
7.3 Replicator sequences include initiator binding
site and easily unwound DNA
The E. coli replicator is called the OriC. It
contains 5 sequences of 9 base pairs (called 9-mers)
that are bound by the initiator DnaA.
Nearby there are 3 sequences called 13-mers that
are A-T rich（easily unwound DNA）.
Fig. 8-25
Initiators have 3 functions:
a) binding to specific
sequences in the
replicator
b) Causing unwinding of the
DNA helix
c) Binding to other proteins
to bring them to the
replicator.
7.4 Protein-protein and protein-DNA interactions
direct the initiation process
oriC复制起点的解
链过程
7.5 Eukaryotic chromosomes are replicated exactly
once per cell cycle.
The initiator in eukaryotic cells is a 6 protein complex
called the origin recognition complex (ORC). Subunits
are named according to their size, with ORC1 being the
largest and ORC6 being the smallest subunit.
ORC recongnizes a conserved sequence A element, as
well as a less- conserved B1 element. 整个细胞周期中
均结合在ARS上.
Binding of ORC does not cause the DNA strands to
unwind.
Binding of ORC does recruit other proteins that are
necessary for replication to the replicator.
Replicators are inactivated
by DNA replication---Whether an origin is activated to cause its own
replication or replicated by a replication for derived
from an adjacent origin, it must be inactivated until
the next round of cell division.
8. Finishing Replication
8.1 Type II DNA topoisomerases are required
to Separate daughter DNA
Topoisomerase IV: a type II DNA
topoisomerase, function to unlink
the interlinked daughter genomes.
8.2 Lagging-strand synthesis is unable to copy the extreme
ends of linear chromosomes
线性双链DNA分子连续复制可能
变短的两个原因：
① 滞后链的最后片段不足以
形成冈畸片段
② 即使足以形成冈畸片段其
引物RNA的切除也能使
DNA链变短
Fig. 8-35
8.3 Telomerase is a novel DNA polymerase that does not
require an exogenous template
Telomerase is different from
other DNA polymerases in that it
does not require a DNA template.
端粒酶由蛋白和RNA组成
Telomerase is a reverse
transcriptase together with a
template RNA
端粒酶延伸G丰富链
Fig. 8-37
8.4 Telomerase solves the end replication problem by
extending the 3’ end of the chromosome
The telomeres only acts on the 3’
end of the telomere. The 5’ end is
accomplished by the laggingstrand DNA replication
machinery.
Fig. 8-38
9. The regulation of genome replication
复制起始后，或是DNA被修饰（甲基化和去甲
基化），或是特异性蛋白因子被修饰（磷酸化
和去磷酸化等），这些修饰使得特异性蛋白因
子对复制起始点的识别状态发生改变。
9.1 E. coli DNA的复制调控：
In E.coli , Dam methylase add methyl group to
the A within every GATC.
The newly synthesized strand is not
methylated by Dam methylase in a few
minutes after the synthesis. ——半甲基化
The SeqAbind thoses methylated.——reduces
the methylated rate and prevents DnaA
from associating with oriC and initiating a
new round of replication.
Reduce the initiation replication
from new copies of oriC
Box 8-7, Fig. 1
原核生物的DNA链延伸的速
度几乎是恒定的（ E.coli
DNA完成复制的时间大约为
40分钟），但细胞生长和增
殖速度取决于培养条件，营
养丰富生长快速。
The mechanisms for prevent rapid
reinitiation do not last until cel division.
Box 8-7, Fig. 1
Re-initiation of bacterial
replication at new origins before
completion of the first round of
replication
生长、增殖速度不同的细胞中，
复制叉的数量不同，快速生长的原
核生物中，复制起始点上可以连续
开始新的DNA复制，表现为，虽然
只有一个复制子，但有多个复制叉，
即一个复制循环没有完成，下一个
复制循环就已经开始。
原核生物DNA的复制与细胞分裂一
般是同步的，但复制与细胞分裂不直
接偶联，复制起始不依赖细胞分裂，
而复制终止则能引发细胞分裂。
Please read the Box 8-7
9.2 真核细胞DNA的复制调控
——Eukaryotic chromosome are replicated
exactly once per cell cycle
真核生物具有多个起始复制子，每
一次DNA复制并不是所有的复制起始 G2
点都需要启动。例如酿酒酵母基因组
约有500个复制子，但每次DNA复制
时只需要启动10－20％的复制起点， S
并且每次DNA复制启动的复制起始点
是随机的。
M
G1
Prereplicative complex formation is the first step in
the initiation of replication in Eukaryotes
The steps for Pre-RC formation:
① The ORC recognize and bind to the
replicator.
② Two (Cdc6 and Cdt1) helicase loading
proteins(解旋酶装卸蛋白)are recruited.
③ Then the eukaryotic replication fork
helicase (真核复制叉解旋酶)Mcm2-7
complex is recruited.
④ Pre-RC formed in G1 phase
Fig. 8-30
pre-replicative
complex (Pre-RC)
Pre-RC formation and activation are regulated to allow
only a single round of replication during each cell cycle
Pre-RC are activated to initiate
replication by two protein kinases
——Cdk(cyclin dependent kinase 细
胞周期依赖性激酶)
——Ddk(Dbf4- dependent kinase )
Each of these kinases is
activated only when the cell
enter S phase
Then the polymerases are
recruited
Fig. 8-33