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MindMap Gallery Biochemistry and Molecular Biology—DNA Synthesis
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Biochemistry and Molecular Biology—DNA Synthesis
People's Medical Publishing House, Ninth Edition "Biochemistry and Molecular Biology" Chapter 12 Synthesis of DNA, including the eukaryotic DNA replication process, the basic laws of DNA replication, the enzymology and topology of DNA replication, etc.
Edited at 2023-11-12 10:56:05
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Biochemistry and Molecular Biology—DNA Synthesis
- bacteria
This is a mind map about bacteria, and its main contents include: overview, morphology, types, structure, reproduction, distribution, application, and expansion. The summary is comprehensive and meticulous, suitable as review materials.
- Plant asexual reproduction
This is a mind map about plant asexual reproduction, and its main contents include: concept, spore reproduction, vegetative reproduction, tissue culture, and buds. The summary is comprehensive and meticulous, suitable as review materials.
- Reproductive development of animals
This is a mind map about the reproductive development of animals, and its main contents include: insects, frogs, birds, sexual reproduction, and asexual reproduction. The summary is comprehensive and meticulous, suitable as review materials.
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- Outline
DNA synthesis
Basic laws of DNA replication
semi-preservative copy
DNA replicates in a semiconservative manner
During replication, the parent double-stranded DNA is unwound into two single strands, each of which serves as a template to synthesize the daughter strand DNA double-strands with complementary sequences according to the rules of base pairing.
significance
Genetic conservatism (stability)
universality of differences
遗传保守性不是绝对的：由于DNA突变和DNA重组普遍存在
The DNA of the offspring retains all the genetic information of the parents, and the base sequences between the DNA of the parents and offspring are highly consistent.
Bidirectional replication
DNA replication proceeds in both directions from the origin
Prokaryotic DNA replication
There is only one origin of replication
Prokaryotic genome is circular DNA
Single point starting bidirectional replication
replication fork
定义
复制中的模板DNA形成两个延伸方向相反的开链区
正在进行复制的双链DNA所形成的Y形区域
组成
头部
已解旋的两条模板单链
正在进行合成的新链
尾部
尚未解旋的DNA模板双链
Copy starts from the starting point and unwinds in both directions
eukaryotic DNA replication
Multi-start bidirectional copy feature
The eukaryotic genome is large and complex, consisting of multiple chromosomes. All chromosomes need to be replicated, and each chromosome has multiple origins.
Each origin produces two replication forks that move in opposite directions.
When replication is complete, the replication forks meet and join
A replicator is a functional unit that independently completes replication and contains an origin of replication.
A region of DNA replication starting from a DNA replication origin is called a replicon
The distance between two adjacent starting points
semi-discontinuous replication
DNA replication occurs in a semi-discontinuous manner
Reason: DNA polymerase can only catalyze the synthesis of DNA strands from the 5' to 3' direction.
leading strand
The daughter strand DNA generated in the melting direction is synthesized continuously (continuous replication)
lagging strand
Non-continuous replication, as the template strand is unwound, primers are generated and synthesized piece by piece from 5′→3′
The way in which the leading strand replicates continuously and the trailing strand replicates discontinuously is called semi-discontinuous replication
The synthesis of two complementary strands is asymmetric
In terms of primer generation and daughter strand elongation, the lagging strand is later than the leading strand.
Okazaki fragment
A new DNA fragment synthesized along the template strand of the lagging strand
Fragments are connected by DNA ligase
去除引物
填补引物留下的空隙
high fidelity
DNA replication with high fidelity
concept
Very low probability of mismatch
reason
absolute fidelity
"Semi-conservative replication" ensures absolute fidelity of information transfer between parent and offspring DNA molecules
Strict base pairing principles
High-fidelity DNA polymerase adheres to strict base-pairing principles
The complex and fine structure of replication forks
Complex and fine structure of replication forks in vivo improves replication accuracy
There is a repair system
Exonuclease activity and proofreading function of DNA polymerase
Repair system after copying
Light resurrection repair
Cut repair
Reorganization and repair
SOS
correct mismatch
Enzymology and topology of DNA replication
DNA replication
反应本质
酶促脱氧核苷酸聚合反应
底物
dNTP
dATP
dGTP
dCTP
dTTP
three phosphate groups
The one closest to ribose is α-P, and the ones outward are β-P and γ-P.
During the polymerization reaction, α-P is connected to the 3’-OH of ribose at the end of the daughter chain.
模板
解开成单链的DNA母链
反应简式
(dNMP)n+dNTP→（dNMP)n+1 + PPi
N代表4中碱基中的任意一种
三个阶段
起始
延伸
终止
DNA polymerase catalyzes the polymerization of deoxynucleotides
DNA polymerase
full name
DNA-dependent DNA polymerase (DNA pol)
Features
Requires single-stranded DNA template
Only has 5’→3’ polymerase activity, no 3’→5’ polymerase activity
It cannot catalyze the de novo synthesis of new DNA strands, but can only catalyze the addition of dNTP to the 3'-OH end of the existing nucleotide chain (needs a nucleic acid primer with 3-OH)
Prokaryotes have at least 5 types of DNA polymerases
DNApolⅠ
Encoded by polA
Mainly plays a role in DNA damage repair
Plays an auxiliary role in semi-preservative replication
Repair and copy
Remove primers
Proofread errors in copying
Fill in the gaps that appear during copying and repairing
Molecular Structure
The secondary structure is dominated by α-helices
It is hydrolyzed into two fragments using a specific protease
snippet
323 amino acid residues
5'→3' exonuclease activity
Large fragment (Klenow fragment)
Laboratory synthesis of DNA, commonly used tool enzymes in molecular biology research
604 amino acid residues
DNA polymerase activity
3'→5' exonuclease activity
DNApol II
encoded by polB
repair
Features
DNA pol II gene mutates, bacteria can still survive
Participate in emergency repair of DNA damage
It has low template specificity and can catalyze nucleotide polymerization even on damaged DNA templates.
DNApol III
encoded by polC
The enzyme that actually catalyzes replication elongation in prokaryotes
Primer synthesis, primary replication elongation polymerase
Molecular Structure
Asymmetric heteropolymer composed of 10 kinds (17) subunits
2 core enzymes (2α, 2ε, 2θ)
composition
α, ε, θ subunits
effect
α
合成DNA前导链和后随链
5'→3'聚合酶活性
ε
3'→5'外切酶活性（复制保真性所必需）α亚基可增强其活性
执行碱基选择功能
θ
可能起组装的作用
维系二聚体
main effect
Synthetic DNA
Has 5'→3' polymerization activity
A sliding clamp composed of 1 pair of β subunits (4β)
effect
Clamp DNA template strand
Make the enzyme slide along the template
γ-complex (clip loading complex)
composition
γ, δ, δ', ψ, χ, 2τ
The two τ subunits interact with one core enzyme respectively, and their flexible linking regions can ensure that the two core enzymes of a holoenzyme molecule at the replication fork can move relatively independently, and are respectively responsible for synthesizing the leading strand and the lagging strand.
effect
Facilitates sliding clamp loading
Promote the assembly of the holoenzyme onto the template
Enhance core enzyme activity
DNApol IV
encoded by dinB
DNApoI V
Encoded by umu'2C
Translesion-synthesizing DNA polymerase
There are 5 common eukaryotic DNA polymerases
DNApol β
Low fidelity, participating in emergency repair copying
DNApolγ
enzyme for mitochondrial DNA replication
Ⅱ
DNApol δ
Followed chain after synthesis
DNApol epsilon
Synthetic leading strand
DNApolα
Primerase activity
Synthetic primers (catalyze the synthesis of RNA strands)
Ⅲ
The concept of polymerase turnover
DNA pol α合成引物，然后迅速被具有连续合成能力的DNA pol ε和DNA pol δ所替换的过程
Base selection and proofreading functions of DNA polymerase
The fidelity of DNA replication relies on at least three mechanisms
遵守严格的碱基配对规律 （半保留复制）
聚合酶在复制延长时对碱基的选择功能
复制出错时有即时校对功能
Replication fidelity relies on correct base selection
The ε subunit performs base selection function
"Mismatch" experiments found that the core subunit ε in DNA pol III is selective for the incorporation of nucleotides
DNA pol III shows different affinities to different configurations of glycosidic bonds, thus achieving its selection function
Trans
cis
The exonuclease activity in the polymerase identifies and corrects mismatched bases during replication.
mismatch repair
Identify and remove mismatched bases during replication and correct replication errors
Base
exonuclease activity
DNA pol I, DNA pol δ and DNA pol ε all have strong 3'→5' exonuclease activity and can perform mismatch proofreading
Enzyme activity that sequentially hydrolyzes nucleotides from one end of the nucleic acid chain is generally directional.
Topological changes in DNA molecules during replication
How to unravel the DNA double strands is key to understanding the replication mechanism
Multiple enzymes are involved in unwinding DNA and stabilizing the single-stranded state
Related proteins (prokaryotic genes) involved in DNA unwinding during prokaryotic replication
DnaA(dnaA)
Identify functional starting point
DnaB(dnaB)
helicase
利用ATP供能
作用于氢键，使得DNA双链解开成为两条单链
Unravel DNA double strands
DnaC (dnaC)
Transport and coordination of DnaB
DnaG (dnaG)
primase
复制起始时催化生成RNA引物的酶
Catalytic RNA primer generation
SSB
single-stranded DNA binding protein
在复制中维持模板处于单链状态并保护单链的完整
Stabilizes unwound single-stranded DNA
topoisomerase
Topoisomerase II/gyrase
unwind supercoil
DNA topoisomerase changes the supercoiled state of DNA
DNA topoisomerase (referred to as topoisomerase)
type
Type I
Not dependent on ATP
Cut off one of the double strands of DNA to prevent the DNA from tangling during unwinding and rotation, and seal the cut at the appropriate time to make the DNA become relaxed.
Type II
Depends on ATP
Cut the DNA double strands in the positive supercoiled state to relax the supercoil, and then use ATP to supply energy. The broken ends of the relaxed DNA are restored one after another under the catalysis of the same enzyme, and the DNA molecule enters the negative supercoiled state.
Type III
The concept of topology
In physics, it refers to the elastic displacement of an object or image while maintaining the original properties of the object.
DNA ligase ligates single-stranded gaps created during replication
DNA ligase
Connect the 3'-OH end of the DNA strand to the 5'-P end of the other DNA strand to form a phosphodiester bond between the two, thus connecting the two adjacent DNA strands into a complete chain.
Consume ATP
Functions and uses
DNA ligase acts as the final junction nick in replication
Okazaki fragment
It also plays a role in sewing gaps in DNA repair, recombination and splicing.
It is one of the important tool enzymes in genetic engineering
prokaryotic DNA replication process
start of replication
unzipping of DNA
Copy has a fixed starting point
Fixed origin of replication in E.coli
ikB
span
245bp
composition
5 sets of tandem repeats consisting of 9 base pairs (9bp)
DnaA binding site
3 sets of tandem repeats consisting of 13 base pairs (13bp)
Rich in AT
Easy to unchain
Requires the participation of multiple proteins
DNAA
structure
homotetramer
Function
Identify the origin of replication
Recognizes and binds to the tandem repeat sequence (AT region) of oriC
Several DnaA proteins approach each other to form a DNA-protein complex structure, prompting the DNA in the AT region to unwind
DNAB
Alias
helicase
Function
Unravel DNA double strands
DNAC
Function
Transport and coordination of DnaB
Helicase, with the cooperation of DnaC protein, binds and moves along the unwinding direction, unwinding the double strands to a length sufficient for replication, and gradually displaces the DnaA protein
DNA topoisomerase required
topoisomerase II
Transformation of DNA supercoils through cutting, rotating and rejoining
Convert positive supercoil to negative supercoil
Primer synthesis and initiation complex formation
The synthesis of primers requires the catalysis of primase
Objective reasons for needing primase
DNA pol does not have the ability to catalyze the formation of phosphodiester bonds between two free dNTPs
Only has the ability to catalyze the polymerization between the 3'-OH end of nucleic acid fragments and dNTPs
primase
Belongs to RNA polymerase
But it is different from RNA polymerase that catalyzes transcription
利福平是转录用RNA pol的特异性抑制剂，而引物酶对利福平不敏感
Can catalyze the polymerization of NTP without the need for 3'-OH end
Catalyze RNA primer synthesis
Primer length ranges from approximately 5 to 10 nucleotides
Initiation complex (initiator)
definition
The structure of the initiation complex composed of helicase DnaB, DnaC, primase and the replication initiation region of DNA
Features
Movement of the protein components of the initiation complex along the DNA chain requires ATP.
DNA chain elongation during replication
enzyme
DNApol III
direction
5'→3'
Features
On the same replication fork, the leading strand replicates before the lagging strand
The leading and lagging strands are elongated by the same DNA pol III enzyme.
The elongation direction and point of the leading strand and lagging strand are both at the catalytic site of the DNA pol III core enzyme
high speed
Termination of replication
excision primer
enzyme
DNApolⅠ
fill vacancy
The later-replicated fragment is extended to fill the primer gap of the first-replicated fragment.
enzyme
DNApolⅠ
connecting incision
enzyme
DNA ligase
eukaryotic DNA replication process
start of replication
The initiation of DNA replication in eukaryotes is basically similar to that in prokaryotes
Features
Replication is sequential
Replicators are activated in groups instead of starting synchronously
DNA with high transcriptional activity is replicated early in S phase
Highly repetitive sequences such as satellite DNA, the centrosome that connects chromosome diploids, and the ends of linear chromosomes that are telomeres are all replicated in the final stage of S phase.
The origin of replication sequence is more complex
The initiation of replication is also the opening of double strands to form a replication fork, the formation of primers and the synthesis of RNA primers.
The detailed mechanism is not yet fully understood
require the participation of these enzymes
DNApol δ
Followed chain after synthesis
DNApol epsilon
Synthetic leading strand
DNApolα
Primerase activity
Synthetic primers (catalyze the synthesis of RNA strands)
Follow strict base pairing rules (semi-conservative replication)
Proliferating cell nuclear antigen (PCNA) plays a key role in replication initiation and elongation
PCNA
结构
It has the same function and similar conformation as the β subunit of E.coli DNA pol III, that is, it forms a closed circular slidable DNA clamp, and PCNA binds to the primer-template strand under the action of RFC.
同源三聚体
功能
类似于β亚基
使DNA pol δ获得持续合成的能力
Movable clip to promote DNA synthesis
促进核小体生成
用途
PCNA的蛋白质水平是检验细胞增殖能力的重要指标
DNA chain elongation during replication
Elongation of eukaryotic DNA replication occurs through DNA polymerase switching
Definition of polymerase conversion
A process in which DNA pol α synthesizes primers and is quickly replaced by DNA pol ε and DNA pol δ, which have the ability to continuously synthesize.
The length of the Okazaki fragment is roughly equal to the number of DNA bases contained in one nucleosome (135bp) or several times that
FEN1 and RNase H are responsible for removing eukaryotic replicating RNA primers
In terms of enzyme catalytic rate, it is much slower than that of prokaryotes
Since eukaryotes replicate with multiple replicators, the overall speed is not slow.
The replication speed is affected by different organ tissues, different developmental stages and different physiological conditions.
Termination of replication
Eukaryotic DNA is assembled into nucleosomes immediately after synthesis
Eukaryotic DNA replication and nucleosome assembly proceed simultaneously. After replication is completed, chromosomes are assembled and transition from the G2 period to the M phase.
The destruction of nucleosomes is only limited to a short area immediately adjacent to the replication fork. The movement of the replication fork causes the nucleosome to be destroyed, but when the replication fork moves forward, nucleosomes are rapidly formed on the daughter strand.
Most of the original histones will be reused, but new histones also need to be synthesized
Telomerase solves the problem of chromosome end replication
chromosome end duplication problem
The last replicated RNA primer on the DNA substrands at both ends of the chromosome is removed, leaving a gap. If the remaining single-stranded parent strand of DNA is not filled into double strands, it will be enzymatically digested by DNase in the nucleus, and the chromosomes will become shorter and shorter after repeated replications (such as some lower organisms)
telomeres
definition
Terminal structure of linear DNA molecules in eukaryotic chromosomes
morphology
The ends of chromosomal DNA swell into granules
Function
Maintain chromosome stability and DNA replication integrity
Features
Rich in multiple repeats of T-G short sequences, and can be folded into secondary structures
telomerase
It has the functions of providing RNA template and catalyzing reverse transcription
composition
telomerase RNA
telomerase cooperating protein 1
telomerase reverse transcriptase
Function
An enzyme responsible for the extension of telomeres in cells is a basic nucleoprotein reverse transcriptase. It can add telomeric DNA to the ends of eukaryotic cell chromosomes, fill in the telomeres lost during DNA replication, and extend telomere repair. , which prevents telomeres from being lost due to cell division and increases the number of cell divisions.
Mechanism of action
crawling model
Textbook P245
personal understanding
Telomerase first extends the parent strand (by reverse transcription), and then uses the parent strand as a template to extend the daughter strand (recruiting DNA pol)
Telomerase activity is not necessarily proportional to telomere length
Eukaryotic chromosomal DNA can only be replicated once per cell cycle
concept
Replication only occurs in phase S and can only be replicated once
The initiation of DNA replication in eukaryotic cells occurs in two steps
Selection of replicator genes
period
Appeared in G1 phase
Features
Each replicating locus in the genome is assembled into a prereplication complex
Activation of replication origin
period
Appears only after S phase
Features
Will activate pre-RC, recruit several replication gene binding proteins and DNA polymerase, and initiate DNA unwinding
consistent with cell cycle progression
The level of cyclin D increases in the late G1 phase, activating CDK (cyclin-dependent kinase) in S phase. The replication permission factor is a substrate of CDK and is necessary to initiate DNA replication. Replication-permitting factors generally cannot enter the nucleus through the nuclear membrane, but they can enter the nucleus at the end of mitosis and before the nuclear membrane is reorganized, and bind to the origin of DNA replication. Waiting for CDK to be stimulated to enter S phase to activate and initiate replication. Once replication is initiated, replication-permitting factors can become inactive or degraded. During other times of the cell cycle, new replication permission factors cannot enter the nucleus, ensuring that only one genome replication can occur during a cell cycle.
Eukaryotic mitochondrial DNA replicates in a D-loop manner
Features
The origin of replication is not at the same site on the double-stranded DNA, and there are differences in the timing of inner and outer loop replication.
mtDNA is prone to mutations and is difficult to repair after damage.
MtDNA mutations are related to natural phenomena such as aging, and are also related to the occurrence of some diseases
enzyme
DNApolγ
reverse transcription
reverse transcriptase
full name
RNA-dependent DNA polymerase
effect
Catalyzes the synthesis of double-stranded DNA using RNA as a template
three activities
RNA-directed DNA polymerase activity
RNase activity
DNA-directed DNA polymerase activity
cofactor
Zinc ions
Three steps of catalytic reaction
Generation of RNA/DNA hybrid double strands
Reverse transcriptase uses viral genomic RNA as a template to catalyze the polymerization of dNTPs to generate DNA complementary strands. The product is an RNA/DNA hybrid double strand.
Hydrolysis of RNA single strand
The RNA in the hybrid double strand is hydrolyzed by the RNase active component of reverse transcriptase
RNase in infected cells can also hydrolyze RNA strands
Generation of complementary strands of DNA
The single-stranded DNA remaining after RNA is broken down is used as a template to synthesize the second complementary strand of DNA catalyzed by reverse transcriptase.
The discovery of reverse transcription developed the central dogma
central theme