Download Chapter11 DNA复制, RNA的代谢

教
案
~ 2007 学年 第一 学期
2006
学 院
教
名 称
研
生命科学学院
室
课 程
名 称
授 课
对 象
授 课
教 师
陈文利
称
副教授
职
教 材
名 称
2006 年 9 月
生物化学
2005 级生物技术专业
现代生物化学
日
授课题目（教学章、节或主题）
：
教学器材
与工具
第十章 D N A 复 制
授课时间
多媒体设施、黑板与
笔
第 15 周一第 41-44
节
教学目的、要求（例如识记、理解、简单应用、综合应用等层次）
：
核酸的组成成分及性质特点，用动画描述核酸的二级结构及主要的性质，要求学生掌握核酸的二
级结构特征及其稳定作用力。
教学内容（包括基本内容、重点、难点）
：
DNA Replication
Background Information
Watson & Crick
General Features
1) Many enzymes and proteins are required
2) Template & dNTPs/Mg 2+ are required
3) Semi-conservative (半保留）
A key experiment designed by M. Meselson and W. F. Stahl （1958）
4) DNA Unwinding is necessary
5) A Primer (引物） with a free 3' -OH group is required
6) Only in the 5¡ä¡ú3¡ädirection
7) Specific Origin of Replication-Ori C and ARS (Autonomously Replicating
Sequence,自主复制序列）
Three Common Features of Replication Origins
8) Bi-directional (With some exceptions)
9) Semi-discontinuous (半不连续）
Replication fork (复制叉）， Leading strand (前导链）, Lagging strand （后随链） and
Okazaki fragments（冈崎片段）
10) Highly processive (进行性), Highly ordered and Extremely accurate
"Molecular Structure of Nucleic Acids:
A Structure for Deoxyribose Nucleic Acid"
(Nature, April 25, 1953. volume 171:737-738.)
"The novel feature of the structure is the manner in which the two chains are held together by
the purine and pyrimidine bases... The (bases) are joined together in pairs, a single base from
one chain being hydrogen-bonded to a single base from the other chain, so that the two lie side
by side...One of the pair must be a purine and the other a pyrimidine for bonding to
occur. ...Only specific pairs of bases can bond together. These pairs are: adenine (purine) with
thymine (pyrimidine), and guanine (purine) with cytosine (pyrimidine)."
"...in other words, if an adenine forms one member of a pair, on either chain, then on these
assumptions the other member must be thymine; similarly for guanine and cytosine. The
sequence of bases on a single chain does not appear to be restricted in any way. However, if
only specific pairs of bases can be formed, it follows that if the sequence of bases on one chain
is given, then the sequence on the other chain is automatically determined."
"...It has not escaped our notice that the specific pairing we have postulated immediately
suggests a possible copying mechanism for the genetic material. The structure itself suggested
that each strand could separate and act as a template for a new strand, therefore doubling the
amount of DNA, yet keeping the genetic information, in the form of the original sequence,
intact. "
DNA Replication
Since DNA replication is semiconservative, therefore the helix must be unwound.
John Cairns (1963) showed that initial unwinding is localized to a region of the bacterial
circular genome, called an “origin” or “ori” for short.
Enzymes and Proteins Involved in DNA Replication
DNA –dependent DNA polymerase (DNA pol, DNA聚合酶）- incorporation of nucleotides
DNA Helicase（DNA解链酶）- promotes strand separation, requires ATP and unwinds ds DNA
at replication fork
Single-stranded DNA binding proteins（ SSB，单链结合蛋白）-keep strands apart, coat
DNA and prevent re-association of strands and stimulate DNA polymerase
Primase（引发酶）- formation of RNA primers
DNA ligase （DNA 连接酶）-joining of Okazaki fragments
Topoisomerase（拓扑异构酶）- release stress of unwinding: relieves stress by breaking and
sealing--otherwise DNA becomes too tightly coiled and stops the replicating fork
The Enzymes responsible for removing RNA primers
Uracil-DNA N-glycosylase （尿嘧啶-DNA-N-糖苷酶）
Telomerase（端聚酶）-maintain telomeric DNA integrity
DNA-dependent DNA polymerases
Common Reaction Equation:
Mg2+
DNA + Primer-OH + dNTP
DNA/Primer-dNMP + PPi
Subsequent hydrolysis of PPi drives the reaction forward Prokaryotic DNA pol
DNA pol I,II,III,IV and V
Eukaryotic DNA pol
E. coli DNA polymerases
Identification
Kornberg and DNA pol I (Kornberg enzyme)
Structure and Function of DNA pol I
A multi-functional enzyme
DNA pol II and DNA pol III
DNA pol IV and DNA pol V
Conclusion
DNA pol III is a major polymerase involved in E. coli chromosome DNA replication
More on Pol I
Why the exonuclease activity?
The 3'-5' exonuclease activity serves a proofreading function
It removes incorrectly matched bases, so that the polymerase can try again
The DNA Polymerase Family
A total of 5 different DNAPs have been reported in E. coli
DNAP I: does 90% of polymerizing activity
DNAP II: functions in DNA repair (proven in 1999)
DNAP III: principal DNA replication enzyme
DNAP IV: functions in DNA repair (discovered in 1999)
DNAP V: functions in DNA repair (discovered in 1999)
DNA Polymerase III
The "real" replicative polymerase in E. coli
It’s fast: up to 1,000 dNTPs added/sec/enzyme
It’s highly processive: >500,000 dNTPs added before dissociating
It’s accurate: makes 1 error in 107 dNTPs added, with proofreading, this gives a final error rate
of 1 in 1010 overall.
Genetic mutant(Ts)
The structure formed by two beta subunits of the E. coli DNA polymerase III . This structure
can clamp a DNA molecule and slide with the core polymerase along the DNA molecule.
Other Enzymes and Proteins Involved in DNA Replication
Helicase: I and II；ATPase
Helicase II is involved in DNA replication
E.coli: dna B蛋白 and Rep蛋白
Werner syndrome (WS) and Helicase mutation
SSB：without any enzymatic activity
Prokaryotic: Act in a cooperative fashion
Eukaryotic: Replication Factor A (RFA)
Primase: A kind of DNA-dependent RNA polymerase
The Enzyme removing primers
Prokaryotic: DNA pol I; Enkaryotic: RNase H (5’-3’ exonuclease activity active only on
RNA-DNA hybrids) or MF1 (5’-3’ exonuclease )
DNA ligase
Prokaryotic: NAD+ ; Eukaryotic and Viral: ATP
Topoisomerase: I,II (E.coli- Gyrase),III, and IV
II and IV are involved in DNA replication
Uracil-DNA N-glycosylase
Removing the mis-incorporated dUMP during DNA replication
Telomease
Specific to eukaryotes; A kind of retro-transcriptase
Details of DNA Replication
Three steps
1) Initiation（起始）
2) Elongation（延伸）
3) Termination and Separation（终止与分离）
DNA replication in E.coli- “èform”
DNA replication in eukaryotes
D-loop replication and Rolling-circle replication
DNA Replication is an Ordered Series of Steps
Find the origin: DnaA (origin recognition protein) + HU
Unwind the helix: DnaB (helicase), DnaC + DnaT (deliver DnaB
to the origin), SSB (keeps helix unwound), DNA Gyrase
facilitates efficient unwinding
Synthesize primers: DnaG (primase) + PriA, PriB,PriC
(assembly and function of the primosome)
Elongate (new strand synthesis): DNAP III holoenzyme
Remove the primers and ligate Okazaki fragments: (DNAP I + Ligase)
Terminate replication: Ter (termination sequence)
+ Tus (termination utilization substance)
Separate Daughter DNAs: DNA Topo IV
Primosome- 引发体
Gyrase- 旋转酶
Eukaryotic DNA Replication
Like E. coli, but more complex
Chromatin and Nucleosome
Multiple origins of replication
DNA replication occurs just at S phase of the cell cycle and is controlled by many proteins
Okazaki fragments are shorter than in Prokaryotes
Replication forks run a slower speed than in Prokaryotes
Two rounds of replication cannot occur at the same time
Telomerase is required
DNA polymerase error rates
Initial pairing error = 1/105
After proofreading = 1/107~1/108
mismatch repair = 1/1010~1/1011
Human genome = 3.2 x 109 bp
~3 errors/replication!
DNA Damage and Repair
DNA is the only biomolecule that is specifically repaired. All others are either degraded or
replaced.
>100 genes participate in various aspects of DNA repair, even in organisms with very small
genomes.
Many, perhaps most, cancers are at least partially attributable to defects in DNA repair.
Types of Damage
Base loss: Depurination (5000 bases per cell per day )
Base modification
1) Deamination: C→U;A→I (100 bases per cell per day)
2) Chemical modification: alkylation (O6-methylguanine)
3) Photodamage (Especially UV): Thymine dimer
and 6-4 photo product
Replication errors: Mismatch
Inter-strand crosslinks
DNA-protein crosslinks
Strand breaks
Types of Repair
Direct repair
Photolyase（光裂解酶） & Guanine Methyl transferase （鸟嘌呤甲基转移酶）
Excision repair
1) Base excision
2) Nucleotide excision
3) Mismatch repair
Damage tolerance
Attempts to minimize the effects of damage that has not been repaired.
SOS repair & Recombinational repair
Direct Repair of T dimer
Features: Only one enzyme is enough
DNA photolyase:
1) Uses energy from light absorption
2) Contains chromophores (light absorbing agents)
3) Action spectrum is blue/near UV light range
4) photolyases are found in bacteria, fungi, plants and many
vertebrates, but not in placental mammals.
Steps in the repair mechanism:
1) Enzyme recognizes and binds to the damage
2) Light absorption by chromophore converts it to an excited
state
3) Chromophore donates an electron to the cyclobutyl dimer
4) Dimer is destabilized and undergoes a series of electron
rearrangements which result in monomeric pyrimidines
SOS Repair
-Error-prone replication
The SOS repair system is induced in response to major damage to the bacterial DNA or in
response to agents which inhibit DNA replication. The system is a complex one with over 20
genes involved. Two of these are the important regulator genes: lexA and recA.
LexA is a repressor（阻遏蛋白） that regulates the expression of all of the other SOS repair
genes, including recA. It also regulates its own synthesis. LexA is a dimer. Each monomer has
a DNA binding domain and a dimerization domain, however, the protein will not bind to DNA
unless it has formed a dimer first. Normally, LexA binds to its operators（操作子） to block
expression of the SOS repair genes.
Recombinational Repair
Also known as post-replication repair, this system permits the cell to tolerate damage
without actually repairing it. It depends on the mechanisms of homologous recombination
（同源重组） to replace a damaged region of DNA that cannot be repaired with a good copy
of the same region.
重点：原核生物 DNA 合成的过程重点掌握原核生物转录的过程，主要损伤类型
难点： DNA 重组修复的理解
教学过程设计（要求阐明对教学基本内容的展开及教学方法与手段的应用、讨论、作业布置）：
利用课件结合板书介绍蛋白质化学的基础知识，了解 D N A 代 谢 的研究新进展，重点掌
握原 核 生 物 D N A 合 成 的 过 程 重点掌握原 核 生 物 D N A 的复制过程，主
要损伤类型，在理解的基础上布置作业，让学生在作业中发现问题提出问题，对于比较难理解
的老师在课堂上再次强调。在教学过程中给学生介绍学习方法及鼓励学生拓展知识。