9,DNA包装成染色体大约压缩了 7倍,请说明计算的依据。
答,依据是,一个核小体的直经是 10nm,由 200个碱基对的 DNA组成,每个碱基对长度为 0.34nm,
一个核小体伸展开来的长度是 70nm,因此,DNA
包装成核小体,大约压缩了 7倍。
非组蛋白是一类不均一的蛋白质，约有
500多种不同的组分。一般说来，所含酸性氨基酸超过碱性氨基酸，故呈酸性，带负电荷。另外,非组蛋白常常是被磷酸化的。
5.1,Types of Mutation
5.2,Mechanism of Mutation
5.3,Mechanism of Repair DNA
Chapter 5
Gene Mutation & Recombination
5.4,Molecular Mechanism of Recombination
= 3x dNt ± n x Amino acid
= 3x dNt Framshift
transition(转换 )
transvertion(颠换 )
5.1,Types of Point mutation
conversion (取代 )
GAA(Glu) → TAA(stop)
GAA(Glu) → AAA(Lys)
GAA(Glu) → GA G(Glu)---Samesense mut.
---Missense mut.
---Nonsense mut.
conversion effect
dNt deletion or insertion
5.2.1,Spontaneous mutation
5.2.1.1,碱基异构式引起 DNA复制过程的错误
a) 碱基异构式
A(amino) A(imino) C(a) C(i)
G(keto) G(enol) G(k) G(e,i)
T(keto) T(enol-2’) or T(enol-4’)
5.2,Mechanism of Mutation
Amino,氨基 imino,亚氨基 Keto,酮基 enol,烯醇基
b) 碱基异构式引起 DNA复制的错配
A(a) T(k) G(k) C(a)
错误配对 G(k) T(e)A(a) C(i)
A(i,anti) A(a,syn) A(i,anti) G(k,syn)
G(e,i,anti) G(k,syn) G(e,i,anti) A(a,syn)
A(i) C(a) G(e) T(k)
正确配对
5.2.1.2,Unequal crossing-over
3’? 5’ editing function mutation
MCE (mismatch correction enzyme)
错配修复功能丧失 突变率升高修复过程是基因突变的重要来源
5.2.1.3.增变基因（ mutator gene ）
w.t,维持遗传的稳定性
mut,随机引起其他各类基因的突变
DNA polymerase 相关基因
错配修复系统的基因
DNA 损伤修复系统基因基因组中某些基因的突变可使整个基因组的突变率明显上升,
这类基因称为增变基因,(能够增加自发突变频率的基因 )
5.2.2,Induced mutation
5.2.2.1,物理诱变
a) 电离辐射诱变
Co60 (χ)( γ) ray
Cs137(χ) (γ) ray
H3 (α) ray
P32,,S35(β) ray
卫星搭载诱变,高真空，强辐射，微重力
(χ)( γ) ray穿透性
（外照射处理）
(α) (β) ray非穿透性
（内标记处理）
dNt电荷及结构改变
b) 非电离辐射 — Ultra Violet light (U.V)
---pyrimidine dimer (TT dimer )is generated by
covalent links between adjacent TT
∧
U.V.… C T T A…
共价键
5.2.2.2,化学诱变
a) 干扰碱基合成的化学诱变剂
6-Mercalto purine
干扰嘌呤合成
SH
5-Amino Uracil
干扰嘧啶合成
O
O
H2N
A(a)
T(k)
5-BrU(k)
G(k)
5-BrU(e)
C(a)
mispairing
mispairing
T(k)
A(a)
5-BrU(k)G(k)
C(e)
5-BrU(e)
A(a) G(k)
G(k) A(a)
Replication error
Incorporation error
b) base analogys leads base mispairing
HNO2 (Nitrous acid NA)
c) Base modifying chemical mutagen
A
C
G
HNO2
deamination
I C
U A
X C
I:次黄嘌呤
U:尿嘧啶
X:黄嘌呤
NH2OH (Hydroxylamine HA)
C(h) A(a)C(a) HA
d) Alkylation agent mutagens
EMS (Ethyl methane sulfonate) CH3— S— O--CH2CH3
O
O
MMS CH3— S— O--CH3
O
O
SM (Sulfur Mustards gas 硫芥子气 ) HS CH2CH2Cl
CH2CH2Cl
e) Mutagen— insertion--framshift
AO (Acridine Orange) 扁平分子
EB (Ethidium Bromide)
-ATTTTTCG -
-TAAAAAGC-
-A TTTCG -
-T AAAGC-
TAO T
-AT EB TTTTCG-
-TA
AO
EB
-ATTTCG -
-TAAAGC-
-ATX’TTTTCG-
-TAX AAAAGC-X AAAAGC-
5.3.1,复制过程中的错配修复机制 (ξ= 10-11)
+ ----- A-----
- ------C---
DNApol (ξ = 10-8)
经第二次校正 ξ = 10-11
M R S
5.3,Mechanism of Repair DNA
Mismatch Repair System
5.3.1.1,Mismatch repair system
DNA polymerase
ligase
dam gene m6A甲基化酶
MCE (mismatch correct enzyme)
3 subunits mutH,L,S
识别新生链中非 m6A的 GATC序列
Scanning 新生链中错配碱基酶切含错配碱基的新生 DNA区段
MCE scanning
DNA中的 GATC(palindromic seq.)
为 m6A甲基化敏感位点平均每 2kb左右有一 GATC seq.
3’-------C----------CTAG----------CTAG---- 5’
5’
-----------T----------GATC----------GATC----3’
3’
------------------------------------------------------- 5’
少 梯度 多（ m6A）新生链
MCE scanning
DNA中的 GATC(palindromic seq.)
为 m6A甲基化敏感位点平均每 2kb左右有一 GATC seq.
3’-------C----------CTAG----------CTAG---- 5’
5’
-----------T----------GATC----------GATC----3’
3’
------------------------------------------------------- 5’
少 梯度 多（ m6A）新生链
MCE scanning
endonuclease
Polymerase
ligase
GATC GATC
CTAG CTAG
T
C
GATC GATC
CTAG CTAG
T
GATC GATC
CTAG CTAG
T
A
5.3.1.2,ung system (尿嘧啶 -N-糖苷酶系统 )
---TAGC---
---ATCG---
---TAGC---
---A CG---U
---TAGC---
ung-aseGCUA
U
---TAGC---
---A CG---
Apurinase
(内切酶 )
---TAGC---
---A
DNApol
ligaseTCG---
phR 471aa
5.3.2,DNA的损伤修复
5.3.2.1,photo reactivation
----TT----
----AA----
----TT----
----AA----
----TT----
----AA----
----TT----
----AA----
Before replication & Error-free
400 nm Blue light & phR gene
(photo-reactivation enzyme)
可见光激活
5.3.2.2,Exision Repair
Before Replication
Error-free
UvrA,B,C gene
Endonucleases
Exonuclease
DNA pol
Ligase
w.t,UvrA+ RecA+
Rec A+ uvr a-
rec a- uvr a-
Rec-A,gene 以某种方式参与 DNA损伤修复
5.3.2.3,Recombinative Repair (strand transfer repair)
After replication repair
Error-prone
RecA,DNA polymerae
ligase genes be needed
U.V 计量
E.coli 存活 %
5.3.2.4,SOS repair (U.V,reactivation or W reactivation)
Jean Weigle
E.coli E.coli E.coli
λ 80 10 100
mut,100% 50% 10%
Damaged DNA of phage be repaired in E.coli A
SOS repair in E,coli have to be induced by U.V,(A & B)
High frequency mutation by SOS repair (Error-prone)
A B C
● DNA 重组活性
● 与 S.S,DNA结合活性
● 少数蛋白的 proteinase活性
RecA-P
RecA-p不表现 proteinase活性能量大量消耗与 S.S,DNA结合激活 RecA-p的 proteinase活性
LexA-p降解
RecA-p高效表达
300 times up
SOS open
当 DNA复制受阻 / DNA damaged
细胞内原少量表达的 RecA-p
修复损伤
当 DNA正常复制时
5.3.2.5,突变的形成
DNA
未经修复 经过修复 / 校正倾向差错修复
(重组修复,SOS）
避免差错修复
(光修复，切补修复 )
形成突变 不形成突变
DNA damaged / mispairing
物理诱变，化学诱变，自发突变突变是在修复过程中形成的（非准确的修复）
死亡突变率降低
5.4.1.Types of Recombination
a) Homologous Recombination
occur between Homo-chromosome / Homo-seq,
sister & non-sister chromatids
transformation,transduction
conjugation,transfection…
large fragment exchange
Recombination site is in hotspot mostly
Recombinase be needed (RecA,BC)
5.4,Molecular Mechanism of Recombination
b) Transposition Recombination
(replication recombination)
Specific transposable genetic element
Independent on homo-sequence between
Tn & target site
Transpotase be needed
Leads to insertion,deletion,inversion,
rearrangement…
c) Site-Dependent
Specific Recombination
Integrase
(Int-ase be needed & not need RecA-p )
Conservative recombination
attP of λ p O p’
attB of E.coli
B O B’
B O P’ P O B’
Int (Integrase)
IHF (Integration host factor)
Xis (Excisionase)
FIS (factor of inversion
stimulation)
5.4.2,Homologous recombination
5.4.2.1,Earlier Two Hypothesises
a) Breakage— rejoining (1930 Darlington)
a b
A B
b) copy-choice (1931 Belling)
a B
A b
DNA replication in S stage? recombination in M stage?
5.4.2.2,Molecular model of homologous recombination
a) gene conversion(基因转换 )
Neuraspora
A x a
a
A
A,a
meiosis
a
a
A
A
a
A
a
A
一条染色体上特定的遗传基因被同源染色体上的等位基因所替代的非相互重组的现象 — 基因转换
A A A A a A a A a
A A A A a A a A a
A a a A a a A a A
A A a A a a A a A
A a a a A A a a A
a a a a A A a a A
a a a a A a A A a
a a a a A a A A a
5/3 3/5 2/6 4/4
b) Molecular model of homologous recombination
Synapsis (联会 ),paired DNA duplexes
RecBC,nicks made in homologous strands
between two DNA
RecA,leads to broken ends move and cross-over
to pair with complement in other duplex
Holliday Intermediate structure
1964,Holliday,R Holliday Intermediate
1965,Whitehouse Polaron Hybrid DNA model
P Q R
p q r
Q; A/T q,C/G
Nicks are sealed
Cross-over point moves
by branch migration
Isomerization
Generate planar molecular
by rotation
P Q R
p q r
Q; A/T q,C/G
R,r
未交换
Q,q
含 C/T 结构
R,r
发生交换
Q,q
含 C/T 结构
Resolution
in two
directions
Heteroduplex
region made
in Q/q (C/T)
交换不是简单发生在 1 bp之间的断裂 ----错接事件
交换涉及两条 D.S,DNA之间的 holliday intermediate,branch
migration,isomerization,resolution 等一系列复杂的过程
交换发生后，其两端的会出现 Pt,Rt = 1:1的正常分离比例
当交换发生在某对等位基因内，其突变位点可能因交叉移动，
而被包括在 heteroduplex region之内，从而引起基因转换
Conclusion:
基因转换 (conversion)发生的机率,随突变位点离 DNA crossover
point的距离增大，而表现逐渐变小的极性梯度的效应
c) Interrelated enzymes & Hotspot of recombination
RecA-p:
40kd monomer 2000 / cell
RecA-p binding with S.S.DNA
ATPase activity
(S.S.DNA— dependent ATPase activity)
RecA-p to 5000 / cell
U.V,Bleomycin(博来霉素 ),
mitomycin(丝裂霉素 )
Rec.A-p
Hydrogen bond reform
heterodulpex
Rec.A-p
Hydrogen bond reform
heterodulpex
Rec B,C,D— P
RecBCD complex 300kd
Frequency of homologous recombination 100 X?Rec-b,c,d mut,
具有解链酶活性 &核酸外切酶活性
nick release S.S,DNA(被 RecA-p结合 )? 使 dsDNA
使 crossover point 沿 D.S.DNA 解旋的方向移动 (migration)
使 heteroduplex region 重新形成螺旋
RecBCD-p特异识别 GCTGGTGGT 序列并在其下游 4-6 bp
处切断 ssDNA
χ (chi) sequence Hotspot of recombination
χ (chi)— sequence 具有 species,gene &genome seq.的特异性
in E.coli genome 1000 chi-seq.
chi RecBCD
4-6bp
OH
RecA-p
DNA解旋 再螺旋
答,依据是,一个核小体的直经是 10nm,由 200个碱基对的 DNA组成,每个碱基对长度为 0.34nm,
一个核小体伸展开来的长度是 70nm,因此,DNA
包装成核小体,大约压缩了 7倍。
非组蛋白是一类不均一的蛋白质，约有
500多种不同的组分。一般说来，所含酸性氨基酸超过碱性氨基酸，故呈酸性，带负电荷。另外,非组蛋白常常是被磷酸化的。
5.1,Types of Mutation
5.2,Mechanism of Mutation
5.3,Mechanism of Repair DNA
Chapter 5
Gene Mutation & Recombination
5.4,Molecular Mechanism of Recombination
= 3x dNt ± n x Amino acid
= 3x dNt Framshift
transition(转换 )
transvertion(颠换 )
5.1,Types of Point mutation
conversion (取代 )
GAA(Glu) → TAA(stop)
GAA(Glu) → AAA(Lys)
GAA(Glu) → GA G(Glu)---Samesense mut.
---Missense mut.
---Nonsense mut.
conversion effect
dNt deletion or insertion
5.2.1,Spontaneous mutation
5.2.1.1,碱基异构式引起 DNA复制过程的错误
a) 碱基异构式
A(amino) A(imino) C(a) C(i)
G(keto) G(enol) G(k) G(e,i)
T(keto) T(enol-2’) or T(enol-4’)
5.2,Mechanism of Mutation
Amino,氨基 imino,亚氨基 Keto,酮基 enol,烯醇基
b) 碱基异构式引起 DNA复制的错配
A(a) T(k) G(k) C(a)
错误配对 G(k) T(e)A(a) C(i)
A(i,anti) A(a,syn) A(i,anti) G(k,syn)
G(e,i,anti) G(k,syn) G(e,i,anti) A(a,syn)
A(i) C(a) G(e) T(k)
正确配对
5.2.1.2,Unequal crossing-over
3’? 5’ editing function mutation
MCE (mismatch correction enzyme)
错配修复功能丧失 突变率升高修复过程是基因突变的重要来源
5.2.1.3.增变基因（ mutator gene ）
w.t,维持遗传的稳定性
mut,随机引起其他各类基因的突变
DNA polymerase 相关基因
错配修复系统的基因
DNA 损伤修复系统基因基因组中某些基因的突变可使整个基因组的突变率明显上升,
这类基因称为增变基因,(能够增加自发突变频率的基因 )
5.2.2,Induced mutation
5.2.2.1,物理诱变
a) 电离辐射诱变
Co60 (χ)( γ) ray
Cs137(χ) (γ) ray
H3 (α) ray
P32,,S35(β) ray
卫星搭载诱变,高真空，强辐射，微重力
(χ)( γ) ray穿透性
（外照射处理）
(α) (β) ray非穿透性
（内标记处理）
dNt电荷及结构改变
b) 非电离辐射 — Ultra Violet light (U.V)
---pyrimidine dimer (TT dimer )is generated by
covalent links between adjacent TT
∧
U.V.… C T T A…
共价键
5.2.2.2,化学诱变
a) 干扰碱基合成的化学诱变剂
6-Mercalto purine
干扰嘌呤合成
SH
5-Amino Uracil
干扰嘧啶合成
O
O
H2N
A(a)
T(k)
5-BrU(k)
G(k)
5-BrU(e)
C(a)
mispairing
mispairing
T(k)
A(a)
5-BrU(k)G(k)
C(e)
5-BrU(e)
A(a) G(k)
G(k) A(a)
Replication error
Incorporation error
b) base analogys leads base mispairing
HNO2 (Nitrous acid NA)
c) Base modifying chemical mutagen
A
C
G
HNO2
deamination
I C
U A
X C
I:次黄嘌呤
U:尿嘧啶
X:黄嘌呤
NH2OH (Hydroxylamine HA)
C(h) A(a)C(a) HA
d) Alkylation agent mutagens
EMS (Ethyl methane sulfonate) CH3— S— O--CH2CH3
O
O
MMS CH3— S— O--CH3
O
O
SM (Sulfur Mustards gas 硫芥子气 ) HS CH2CH2Cl
CH2CH2Cl
e) Mutagen— insertion--framshift
AO (Acridine Orange) 扁平分子
EB (Ethidium Bromide)
-ATTTTTCG -
-TAAAAAGC-
-A TTTCG -
-T AAAGC-
TAO T
-AT EB TTTTCG-
-TA
AO
EB
-ATTTCG -
-TAAAGC-
-ATX’TTTTCG-
-TAX AAAAGC-X AAAAGC-
5.3.1,复制过程中的错配修复机制 (ξ= 10-11)
+ ----- A-----
- ------C---
DNApol (ξ = 10-8)
经第二次校正 ξ = 10-11
M R S
5.3,Mechanism of Repair DNA
Mismatch Repair System
5.3.1.1,Mismatch repair system
DNA polymerase
ligase
dam gene m6A甲基化酶
MCE (mismatch correct enzyme)
3 subunits mutH,L,S
识别新生链中非 m6A的 GATC序列
Scanning 新生链中错配碱基酶切含错配碱基的新生 DNA区段
MCE scanning
DNA中的 GATC(palindromic seq.)
为 m6A甲基化敏感位点平均每 2kb左右有一 GATC seq.
3’-------C----------CTAG----------CTAG---- 5’
5’
-----------T----------GATC----------GATC----3’
3’
------------------------------------------------------- 5’
少 梯度 多（ m6A）新生链
MCE scanning
DNA中的 GATC(palindromic seq.)
为 m6A甲基化敏感位点平均每 2kb左右有一 GATC seq.
3’-------C----------CTAG----------CTAG---- 5’
5’
-----------T----------GATC----------GATC----3’
3’
------------------------------------------------------- 5’
少 梯度 多（ m6A）新生链
MCE scanning
endonuclease
Polymerase
ligase
GATC GATC
CTAG CTAG
T
C
GATC GATC
CTAG CTAG
T
GATC GATC
CTAG CTAG
T
A
5.3.1.2,ung system (尿嘧啶 -N-糖苷酶系统 )
---TAGC---
---ATCG---
---TAGC---
---A CG---U
---TAGC---
ung-aseGCUA
U
---TAGC---
---A CG---
Apurinase
(内切酶 )
---TAGC---
---A
DNApol
ligaseTCG---
phR 471aa
5.3.2,DNA的损伤修复
5.3.2.1,photo reactivation
----TT----
----AA----
----TT----
----AA----
----TT----
----AA----
----TT----
----AA----
Before replication & Error-free
400 nm Blue light & phR gene
(photo-reactivation enzyme)
可见光激活
5.3.2.2,Exision Repair
Before Replication
Error-free
UvrA,B,C gene
Endonucleases
Exonuclease
DNA pol
Ligase
w.t,UvrA+ RecA+
Rec A+ uvr a-
rec a- uvr a-
Rec-A,gene 以某种方式参与 DNA损伤修复
5.3.2.3,Recombinative Repair (strand transfer repair)
After replication repair
Error-prone
RecA,DNA polymerae
ligase genes be needed
U.V 计量
E.coli 存活 %
5.3.2.4,SOS repair (U.V,reactivation or W reactivation)
Jean Weigle
E.coli E.coli E.coli
λ 80 10 100
mut,100% 50% 10%
Damaged DNA of phage be repaired in E.coli A
SOS repair in E,coli have to be induced by U.V,(A & B)
High frequency mutation by SOS repair (Error-prone)
A B C
● DNA 重组活性
● 与 S.S,DNA结合活性
● 少数蛋白的 proteinase活性
RecA-P
RecA-p不表现 proteinase活性能量大量消耗与 S.S,DNA结合激活 RecA-p的 proteinase活性
LexA-p降解
RecA-p高效表达
300 times up
SOS open
当 DNA复制受阻 / DNA damaged
细胞内原少量表达的 RecA-p
修复损伤
当 DNA正常复制时
5.3.2.5,突变的形成
DNA
未经修复 经过修复 / 校正倾向差错修复
(重组修复,SOS）
避免差错修复
(光修复，切补修复 )
形成突变 不形成突变
DNA damaged / mispairing
物理诱变，化学诱变，自发突变突变是在修复过程中形成的（非准确的修复）
死亡突变率降低
5.4.1.Types of Recombination
a) Homologous Recombination
occur between Homo-chromosome / Homo-seq,
sister & non-sister chromatids
transformation,transduction
conjugation,transfection…
large fragment exchange
Recombination site is in hotspot mostly
Recombinase be needed (RecA,BC)
5.4,Molecular Mechanism of Recombination
b) Transposition Recombination
(replication recombination)
Specific transposable genetic element
Independent on homo-sequence between
Tn & target site
Transpotase be needed
Leads to insertion,deletion,inversion,
rearrangement…
c) Site-Dependent
Specific Recombination
Integrase
(Int-ase be needed & not need RecA-p )
Conservative recombination
attP of λ p O p’
attB of E.coli
B O B’
B O P’ P O B’
Int (Integrase)
IHF (Integration host factor)
Xis (Excisionase)
FIS (factor of inversion
stimulation)
5.4.2,Homologous recombination
5.4.2.1,Earlier Two Hypothesises
a) Breakage— rejoining (1930 Darlington)
a b
A B
b) copy-choice (1931 Belling)
a B
A b
DNA replication in S stage? recombination in M stage?
5.4.2.2,Molecular model of homologous recombination
a) gene conversion(基因转换 )
Neuraspora
A x a
a
A
A,a
meiosis
a
a
A
A
a
A
a
A
一条染色体上特定的遗传基因被同源染色体上的等位基因所替代的非相互重组的现象 — 基因转换
A A A A a A a A a
A A A A a A a A a
A a a A a a A a A
A A a A a a A a A
A a a a A A a a A
a a a a A A a a A
a a a a A a A A a
a a a a A a A A a
5/3 3/5 2/6 4/4
b) Molecular model of homologous recombination
Synapsis (联会 ),paired DNA duplexes
RecBC,nicks made in homologous strands
between two DNA
RecA,leads to broken ends move and cross-over
to pair with complement in other duplex
Holliday Intermediate structure
1964,Holliday,R Holliday Intermediate
1965,Whitehouse Polaron Hybrid DNA model
P Q R
p q r
Q; A/T q,C/G
Nicks are sealed
Cross-over point moves
by branch migration
Isomerization
Generate planar molecular
by rotation
P Q R
p q r
Q; A/T q,C/G
R,r
未交换
Q,q
含 C/T 结构
R,r
发生交换
Q,q
含 C/T 结构
Resolution
in two
directions
Heteroduplex
region made
in Q/q (C/T)
交换不是简单发生在 1 bp之间的断裂 ----错接事件
交换涉及两条 D.S,DNA之间的 holliday intermediate,branch
migration,isomerization,resolution 等一系列复杂的过程
交换发生后，其两端的会出现 Pt,Rt = 1:1的正常分离比例
当交换发生在某对等位基因内，其突变位点可能因交叉移动，
而被包括在 heteroduplex region之内，从而引起基因转换
Conclusion:
基因转换 (conversion)发生的机率,随突变位点离 DNA crossover
point的距离增大，而表现逐渐变小的极性梯度的效应
c) Interrelated enzymes & Hotspot of recombination
RecA-p:
40kd monomer 2000 / cell
RecA-p binding with S.S.DNA
ATPase activity
(S.S.DNA— dependent ATPase activity)
RecA-p to 5000 / cell
U.V,Bleomycin(博来霉素 ),
mitomycin(丝裂霉素 )
Rec.A-p
Hydrogen bond reform
heterodulpex
Rec.A-p
Hydrogen bond reform
heterodulpex
Rec B,C,D— P
RecBCD complex 300kd
Frequency of homologous recombination 100 X?Rec-b,c,d mut,
具有解链酶活性 &核酸外切酶活性
nick release S.S,DNA(被 RecA-p结合 )? 使 dsDNA
使 crossover point 沿 D.S.DNA 解旋的方向移动 (migration)
使 heteroduplex region 重新形成螺旋
RecBCD-p特异识别 GCTGGTGGT 序列并在其下游 4-6 bp
处切断 ssDNA
χ (chi) sequence Hotspot of recombination
χ (chi)— sequence 具有 species,gene &genome seq.的特异性
in E.coli genome 1000 chi-seq.
chi RecBCD
4-6bp
OH
RecA-p
DNA解旋 再螺旋
