参与原核生物DNA复制的
酶类和蛋白质
Enzymes and Proteins Involved in DNA
Replication in Prokaryotes
高方远 马欣荣 康海岐
DNA replication(bacteria)
Initiation
Elongation
Termination
Daughter DNA partition
Origins
* the origin of replication is defined
* replication bubble.
* Replication fork
* Unidirectional or bidirectional.
Elongation(semidiscontinuous)
Figure 13.9 The leading strand is synthesized
continuously while the lagging strand is synthesized
discontinuously.
termination
参与DNA复制的酶类
1、DNA聚合酶
2、DNA引发酶（DNA primase）
3、DNA连接酶
4、与DNA几何学性质相关的酶
DNA polymerases in E.coli
I
polA major repair enzyme
II
polB minor repair enzyme
III polC replicase
IV dinB SOS repair
V
umuC、D SOS repair
DNA Polymerase I
DNA polymerase
3’
5’ exonuclease
5’
3’ exonuclease
Figure 13.8 The catalytic domain of a DNA
polymerase has a DNA-binding cleft created
by three subdomains. The active site is in the
palm. Proofreading is provided by a separate
active site in an exonuclease domain.
Figure 13.7 Crystal
structure of phage
T7 DNA
polymerase has a
right hand
structure. DNA lies
across the palm
and is held by the
fingers and thumb.
Photograph kindly
provided by
Charles
Richardson and
Tom Ellenberger.
DNA Polymerase I
Figure 13.5 Nick translation replaces part of a preexisting strand of duplex DNA with newly
synthesized material.
Subunit composition of E.coli DNA polymerase III holoenzyme
subunit
α
ε
θ
ι
γ
δ
δ’
χ
ψ
β
molecular mass
(KDa)
129.9
27.5
8.6
71.1
47.5
38.7
36.9
16.6
15.2
40.6
function
subassemblies
DNA polymerase
3’
5’ exonuclease
core
stimulates exonuclease
dimerizes core
Pol III’
binds γ complex
binds ATP
binds to β
binds to γ and δ
γ complex
binds to SSB
DNA-dependent
binds to χ and γ
ATPase
sliding clamp
Pol III
E.coli Pol III Beta-subunit
Figure 13.18 DNA
polymerase III
holoenzyme assembles in
stages, generating an
enzyme complex that
synthesizes the DNA of
both new strands.
Fig. 1. Model of SOS
translesion replication by DNA
polymerase V. The two DNA
strands are shown as green lines,
and the replication-blocking
lesion is represented by the red
rectangle. The three major steps
in TLR are pre-initiation (2), in
which the RecA nucleoprotein
filaments assembles; initiation
(3 and 4), which involves
binding of pol V to the primertemplate and loading of
the subunit clamp; and lesion
bypass by pol V holoenzyme (5).
SSB is suggested to help in
displacing RecA from DNA both
at the initiation and lesion
bypass steps.
E. coli DNA polymerase IV
（ dinB gene ）
* dinB 基因的表达需要 DNA损伤诱导
* 与UmuC、 UmuD同属Y 家族DNA聚合酶
* E. coli DNA polymerase IV无校正功能，易于延长一些凸
出的引物或模板结构。
2、DNA引发酶（DNA primase）
• Use host RNA polymerase as primase (M13)
• primosome primase (dnaG protein)
(E.coli)
other proteins
фX174: only primase, without the other
proteins
Initiation requires
several enzymatic
activities, including
helicases, single-strand
binding proteins, and
synthesis of the primer.
•Adenovirus terminal
protein binds to the 5
end of DNA and
provides a C-OH end to
prime synthesis of a new
DNA strand.
DNA Polymerase I
A primer terminus is generated within duplex DNA.
Nick translation replaces part of a pre-existing
strand of duplex DNA with newly synthesized
material.
与DNA几何学性质相关的酶
解旋酶（Helicase）
拓扑异构酶（Topoisomerases）
解旋酶（Helicase）
至少4种helicases
* rep helicase
* DNA helicase II
* DNA helicase III
* dnaB Protein: 在E.coli DNA复制中解开
DNA双链
拓扑异构酶（Topoisomerases）
拓扑异构酶I(topA gene)
act on highly negatively
supercoiled DNA
stabilize single-stranded regions
Figure 14.16 Bacterial
type I topoisomerases
recognize partially
unwound segments of
DNA and pass one strand
through a break made in
the other.
拓扑异构酶II
Type II topoisomerases generally relax
both negative and positive supercoils.
The reaction requires ATP
Figure 14.17 Type II
topoisomerases can pass a
duplex DNA through a
double-strand break in
another duplex.
拓扑异构酶IV
与子代DNA分子的分开有关
参与DNA复制的蛋白质
1、参与复制起始的蛋白质因子
Original complx: DnaA、DnaB、DnaC、 DnaG、
HUand SSB
The minimal origin is defined by the
distance between the outside members of
the 13-mer and 9-mer repeats
Prepriming involves
formation of a complex
by sequential association
of proteins, leading to
the separation of DNA
strands.
methylation at the origin
SeqA
A membrane-bound inhibitor binds to
hemimethylated DNA at the origin, and may
function by preventing the binding of DnaA. It is
released when the DNA is remethylated.
The complex at oriC
can be detected by
electron microscopy.
Antibodies
of dnaA
protein HU
The protein HU is a general DNA-binding protein in E. coli .
Its presence is not absolutely required to initiate replication in
vitro, but it stimulates the reaction. HU has the capacity to
bend DNA, and is likely to be involved in some general
structural capacity.
2、参与复制延伸的蛋白质因子
(DnaB)
(Dna G)
2、
参与
复制
终止
的蛋
白质
因子
Tus
How do the daughter DNAs
become disentangled?
与真核生物不同，细菌的DNA复制、染色体重
新折叠以及姊妹染色体的分开是同时发生的。
细菌中姊妹染色体的分开的机制与真核生物不
同。细菌染色体的DNA分子本身卷入了分开机制。
细菌的多复制叉复制（multiple replication）
与真核生物的复制方式不同。
*
多拷贝的oriC
*
只有一个终止序列
SMC类似物
A simplified model of the bacterial cell cycle.
The model is simplified to ignore multifork replication.
A model of a circular chromosome that is undergoing multifork
replication in a rod-shaped bacterium.
复制起点及终点在细胞中的位置
1、膜片段中有复制起始区与终止区的富集推断锚定
蛋白在定位中的作用。
SeqA
2、位于中间位置 复制工厂 的动力作用
多蛋白复合体：polymerase, helicase and accociated
proteins
特殊蛋白质（PolC-GFP、SeqA) 的定位；H3同位素标记；
pull DNA template
duplicated
release DNA outward during replication
The extrusion-capture
model for
bacterial chromosome
partitioning.
3、与染色体组织（organization）、紧结
(compaction)和超螺旋(helicase)有关的蛋白质作用
SMC
Partitioning Motor protein
MukB
(altered)
to organize the chromosome into a higher
order structure by constraining supercoils.(cause)
Chromosome partitioning(consequence)
HU; Hbsu ;
E.coli
Tyrosine site-specific
recombinases
CodV, RipX
B.subtilis
FtsK
Terminusspecific
chromosome
partitioning
events.
Post-septation
partitioning
PBP2 RodA
PBP 3
EnvA
peptidoglycan(肽聚糖)
Figure 12.27 Failure of
cell division generates
multinucleated filaments.
Minicells: anucleate cells
E. coli generate
anucleate cells when
chromosome segregation
fails. Cells with
chromosomes stain blue;
daughter cells lacking
chromosomes have no
blue stain. This field
shows cells of the mukB
mutant; both normal
and abnormal divisions
can be seen. Photograph
kindly provided by Sota
Hiraga.
Problem
1. 哪些DNA位点和蛋白质对复制起点的移动及定位起决定性作
用？
2. 这些位点和蛋白质在指数生长期和进入静止期是否相同？
3. 细胞分裂后，终止序列是怎样定位到细胞的中央部位的？
4. 能够进行多复制叉复制的细菌与不能进行多复制叉复制的细
菌在染色体分开机制上有什么不同？
5. 不同物理形状的染色体分开时有差异吗？
20.