大学分子生物学经典双语课件C3： DNA replication
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Chapter 03: DNA Replication
3.1 The principle of DNA replication 3.2 DNA replication model 3.3 Enzymes and protein needed in DNA replication 3.4 Process of DNA replication 3.5 Telomere and Telomerase
parental duplex is unwound.
On the lagging strand, a stretch of single-stranded
parental DNA must be exposed, and then a
segment is synthesized in the reverse direction (relative to fork movement). A series of these fragments are synthesized, then they are joined together to create an intact lagging strand.
v33enzymesandproteinsneededindnareplicationdna聚合酶dna聚合酶dna聚合酶结构基因polapolbpolc亚基1410相对分子质量1030008800083000053聚合酶活性是是是35外切酶活性校正是是是53外切酶活性是否否聚合速度ntss1620402501000持续合成能力32001500500000功能切除引物修复修复复制表31大肠杆菌dna聚合酶的比较?53exonucleaseactivity
enters newly synthesized DNA in the form of
short fragments(~1000-2000 bases)
The ligase temperature-sensitive mutants → under the temperature in which the ligase is inactive → a large amount of short fragments but
3.1.2 Semi-discontinuous replication
3’
OK !
5’
5’
3’
5’
3’
Replication fork
3’
5’
How ?
1968 Okazaki radioactive label (3H-dTTP) + CsCl density gradient centrifugation → label
Okazaki fragments: the short stretches of 1000-
2000 bases in prokaryotes (100-200 bases in
eukaryotes) produced during semi-discontinuous replication, they are later joined into a covalently intact strand.
3.2.1 θ form
Carins E.coli DNA
3.2.2 Rolling circle form
A
B
3.2.3 D Loop form
3.3 Enzymes and proteins needed in DNA replication 3.3.1 Enzymes and proteins in pro DNA replication 3.3.1.1 DNA polymerases（DNA pols）
called a primase synthesizes an RNA chain(primer)
that provides the priming end.
ssDNA virus
RF（replicating form）
M13
+
expression, packaging, release
M13 No M13 RF Rifampin M13 RF M13 Mme begin bidirectional replication from the unique OriC .There is a single termination site roughly 180o opposite the only origin.
The long, linear DNA molecules of eukaryotic chromosomes consist of multiple replicons, each with its own origin. When replication forks from adjacent replication bubbles meet, they fuse to form the completely replicated DNA. No distinct termini are required.
Rifampin E.coli
[ Rif S ]
E.coli
[ Rif S ] + M13
Rifampin E.coli
[ Rif
R]
• Rifampin is inhibitor of E.coli RNA polymerase
Conclusion
• It’s necessary of RNA polymerase to synthesize a
RNA in the formation of M13 RF；
• after the initiation of M13 RF, the inhibition of rifampin is invalid
The first evidence supporting RNA primer
Purpose: keep the high "faithfulness"
3.1 The principle of DNA replication
3.1.1 semi-conservative replication
Definition: during replication, the two parental strands separate and each acts as a template to direct the synthesis of a new complementary
There are at least 5 kinds of DNA pols in E.coli;
Replication---- DNA pol III
Delete primer---- DNA pol I
Repair--- DNA pol II;IV;V
表3-1 大肠杆菌DNA聚合酶的比较
3.2 Replication Model
Replicon：means a sequence from a origin to a terminus. Origins tend to be A· T-rich to make opening easier
All prokaryotic chromosomes and many phage and viral DNA molecules are circlular and comprise single replicon.
Nick translation: DNA polymerase I has the
ability to start replication in vitro at a nick in DNA. At a point where a phosphodiester bond has been broken in a double-stranded DNA, the enzyme extends the 3’-OH end. As the new
Semi-discontinuous replication-- the leading strand is synthesized continuously while the lagging strand is synthesized discontinuously
3.1.3 Primer
A common feature of all DNA polymerases is that
they cannot initiate synthesis of a DNA chain de
novo.
All DNA polymerases require a 3’-OH end to initiate DNA synthesis. For DNA replication, a special RNA polymerase
daughter strand.
DNA replication pattern: conservative model、
semi-conservative model、dispersive model
E.coli ：Radioactive isotope(15N) label
CsCl density gradient centrifugation
no lagging strand
Conclusion: The lagging strand must be synthesized in the form of Okazaki fragments
On the leading strand, DNA synthesis can proceed continuously in the 5’ to 3’ direction as the
Results: improve the accuracy of the DNA replication up to 105 times.
3.1.4 Direction
Unidirectional Bidirectional
(more common)
Gyurasits and Wake
Autoradiography, Bacillus sp.
DNA 聚合酶Ⅰ 结构基因 亚基 相对分子质量 5′ →3′ 聚合酶活性 3′ →5′ 外切酶活性（校正） 5′ →3′ 外切酶活性 聚合速度（nts/s） 持续合成能力 功能 polA 1 103 000 是 是 是 16~20 3~200 切除引物，修复 DNA 聚合酶Ⅱ polB ≥4 88 000 是 是 否 40 1 500 修复 DNA 聚合酶Ⅲ polC ≥10 830 000 是 是 否 250~1 000 ≥500 000 复制
DNA polymerase I
5’ → 3’ exonuclease activity：removing RNA primer 3’→5’ exonuclease activity: is used to excise bases that have been added to DNA incorrectly — proofreading；
Reason: DNA polymerase has proofreading
activity, while RNA polymerase don't. After
the low-fidelity primer complete its function, it
will be replaced by high-fidelity DNA synthesized DNA polymerase.
Klenow fragment: The larger cleavage product (68 kD) of DNA polⅠby proteolytic treatment (Bacillus subtilis protease), lacking 5’→3’ exonuclease activity；
Importance: the stability of DNA metabolism
The
stability is relative, while the variation is absolute
physical and chemical mutagens → DNA damage； During replication or transcription → base mismatch； During growth and development →modify, deletion, recombination
3.1 The principle of DNA replication 3.2 DNA replication model 3.3 Enzymes and protein needed in DNA replication 3.4 Process of DNA replication 3.5 Telomere and Telomerase
parental duplex is unwound.
On the lagging strand, a stretch of single-stranded
parental DNA must be exposed, and then a
segment is synthesized in the reverse direction (relative to fork movement). A series of these fragments are synthesized, then they are joined together to create an intact lagging strand.
v33enzymesandproteinsneededindnareplicationdna聚合酶dna聚合酶dna聚合酶结构基因polapolbpolc亚基1410相对分子质量1030008800083000053聚合酶活性是是是35外切酶活性校正是是是53外切酶活性是否否聚合速度ntss1620402501000持续合成能力32001500500000功能切除引物修复修复复制表31大肠杆菌dna聚合酶的比较?53exonucleaseactivity
enters newly synthesized DNA in the form of
short fragments(~1000-2000 bases)
The ligase temperature-sensitive mutants → under the temperature in which the ligase is inactive → a large amount of short fragments but
3.1.2 Semi-discontinuous replication
3’
OK !
5’
5’
3’
5’
3’
Replication fork
3’
5’
How ?
1968 Okazaki radioactive label (3H-dTTP) + CsCl density gradient centrifugation → label
Okazaki fragments: the short stretches of 1000-
2000 bases in prokaryotes (100-200 bases in
eukaryotes) produced during semi-discontinuous replication, they are later joined into a covalently intact strand.
3.2.1 θ form
Carins E.coli DNA
3.2.2 Rolling circle form
A
B
3.2.3 D Loop form
3.3 Enzymes and proteins needed in DNA replication 3.3.1 Enzymes and proteins in pro DNA replication 3.3.1.1 DNA polymerases（DNA pols）
called a primase synthesizes an RNA chain(primer)
that provides the priming end.
ssDNA virus
RF（replicating form）
M13
+
expression, packaging, release
M13 No M13 RF Rifampin M13 RF M13 Mme begin bidirectional replication from the unique OriC .There is a single termination site roughly 180o opposite the only origin.
The long, linear DNA molecules of eukaryotic chromosomes consist of multiple replicons, each with its own origin. When replication forks from adjacent replication bubbles meet, they fuse to form the completely replicated DNA. No distinct termini are required.
Rifampin E.coli
[ Rif S ]
E.coli
[ Rif S ] + M13
Rifampin E.coli
[ Rif
R]
• Rifampin is inhibitor of E.coli RNA polymerase
Conclusion
• It’s necessary of RNA polymerase to synthesize a
RNA in the formation of M13 RF；
• after the initiation of M13 RF, the inhibition of rifampin is invalid
The first evidence supporting RNA primer
Purpose: keep the high "faithfulness"
3.1 The principle of DNA replication
3.1.1 semi-conservative replication
Definition: during replication, the two parental strands separate and each acts as a template to direct the synthesis of a new complementary
There are at least 5 kinds of DNA pols in E.coli;
Replication---- DNA pol III
Delete primer---- DNA pol I
Repair--- DNA pol II;IV;V
表3-1 大肠杆菌DNA聚合酶的比较
3.2 Replication Model
Replicon：means a sequence from a origin to a terminus. Origins tend to be A· T-rich to make opening easier
All prokaryotic chromosomes and many phage and viral DNA molecules are circlular and comprise single replicon.
Nick translation: DNA polymerase I has the
ability to start replication in vitro at a nick in DNA. At a point where a phosphodiester bond has been broken in a double-stranded DNA, the enzyme extends the 3’-OH end. As the new
Semi-discontinuous replication-- the leading strand is synthesized continuously while the lagging strand is synthesized discontinuously
3.1.3 Primer
A common feature of all DNA polymerases is that
they cannot initiate synthesis of a DNA chain de
novo.
All DNA polymerases require a 3’-OH end to initiate DNA synthesis. For DNA replication, a special RNA polymerase
daughter strand.
DNA replication pattern: conservative model、
semi-conservative model、dispersive model
E.coli ：Radioactive isotope(15N) label
CsCl density gradient centrifugation
no lagging strand
Conclusion: The lagging strand must be synthesized in the form of Okazaki fragments
On the leading strand, DNA synthesis can proceed continuously in the 5’ to 3’ direction as the
Results: improve the accuracy of the DNA replication up to 105 times.
3.1.4 Direction
Unidirectional Bidirectional
(more common)
Gyurasits and Wake
Autoradiography, Bacillus sp.
DNA 聚合酶Ⅰ 结构基因 亚基 相对分子质量 5′ →3′ 聚合酶活性 3′ →5′ 外切酶活性（校正） 5′ →3′ 外切酶活性 聚合速度（nts/s） 持续合成能力 功能 polA 1 103 000 是 是 是 16~20 3~200 切除引物，修复 DNA 聚合酶Ⅱ polB ≥4 88 000 是 是 否 40 1 500 修复 DNA 聚合酶Ⅲ polC ≥10 830 000 是 是 否 250~1 000 ≥500 000 复制
DNA polymerase I
5’ → 3’ exonuclease activity：removing RNA primer 3’→5’ exonuclease activity: is used to excise bases that have been added to DNA incorrectly — proofreading；
Reason: DNA polymerase has proofreading
activity, while RNA polymerase don't. After
the low-fidelity primer complete its function, it
will be replaced by high-fidelity DNA synthesized DNA polymerase.
Klenow fragment: The larger cleavage product (68 kD) of DNA polⅠby proteolytic treatment (Bacillus subtilis protease), lacking 5’→3’ exonuclease activity；
Importance: the stability of DNA metabolism
The
stability is relative, while the variation is absolute
physical and chemical mutagens → DNA damage； During replication or transcription → base mismatch； During growth and development →modify, deletion, recombination