Part 3 Genetic Information Transfer
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Transcript of Part 3 Genetic Information Transfer
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Part 3
Genetic Information Transfer
The biochemistry and molecular biology department of CMU
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DNA RNA protein
transcription translation replication
reverse transcription
Central dogma
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• Replication: synthesis of daughter DNA from parental DNA
• Transcription: synthesis of RNA using DNA as the template
• Translation: protein synthesis using mRNA molecules as the template
• Reverse transcription: synthesis of DNA using RNA as the template
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Chapter 10
DNA Replication
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Section 1
General Concepts of DNA Replication
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DNA replication
• A reaction in which daughter DNAs are synthesized using the parental DNAs as the template.
• Transferring the genetic information to the descendant generation with a high fidelity
replication
parental DNAdaughter DNA
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Daughter strand synthesis
• Chemical formulation:
• The nature of DNA replication is a series of 3´- 5´phosphodiester bond formation catalyzed by a group of enzymes.
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Phosphodiester bond formation
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Template: double stranded DNA
Substrate: dNTP
Primer: short RNA fragment with a free 3´-OH end
Enzyme: DNA-dependent DNA polymerase (DDDP),
other enzymes,
protein factor
DNA replication system
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Characteristics of replication
Semi-conservative replication
Bidirectional replication
Semi-continuous replication
High fidelity
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§1.1 Semi-Conservative Replication
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Semiconservative replication
Half of the parental DNA molecule is conserved in each new double helix, paired with a newly synthesized complementary strand. This is called semiconservative replication
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Semiconservative replication
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Experiment of DNA semiconservative replication
"Heavy" DNA(15N)
grow in 14N medium
The first generation
grow in 14N medium
The second generation
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Significance
The genetic information is ensured to be transferred from one generation to the next generation with a high fidelity.
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§1.2 Bidirectional Replication
• Replication starts from unwinding the dsDNA at a particular point (called origin), followed by the synthesis on each strand.
• The parental dsDNA and two newly formed dsDNA form a Y-shape structure called replication fork.
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3'
5'
5'
3'
5'
3'
5'3'
direction of replication
Replication fork
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Bidirectional replication
• Once the dsDNA is opened at the origin, two replication forks are formed spontaneously.
• These two replication forks move in opposite directions as the syntheses continue.
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Bidirectional replication
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Replication of prokaryotes
The replication process starts from the origin, and proceeds in two opposite directions. It is named replication.
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Replication of eukaryotes
• Chromosomes of eukaryotes have multiple origins.
• The space between two adjacent origins is called the replicon, a functional unit of replication.
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origins of DNA replication (every ~150 kb)
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§1.3 Semi-continuous Replication
The daughter strands on two template strands are synthesized differently since the replication process obeys the principle that DNA is synthesized from the 5´ end to the 3´end.
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5'
3'
3'
5'
5'
direction of unwinding3'
On the template having the 3´- end, the daughter strand is synthesized continuously in the 5’-3’ direction. This strand is referred to as the leading strand.
Leading strand
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Semi-continuous replication
3'
5'
5'3'
replication direction
Okazaki fragment
3'
5'
leading strand
3'
5'
3'
5'replication fork
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• Many DNA fragments are synthesized sequentially on the DNA template strand having the 5´- end. These DNA fragments are called Okazaki fragments. They are 1000 – 2000 nt long for prokaryotes and 100-150 nt long for eukaryotes.
• The daughter strand consisting of Okazaki fragments is called the lagging strand.
Okazaki fragments
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Continuous synthesis of the leading strand and discontinuous synthesis of the lagging strand represent a unique feature of DNA replication. It is referred to as the semi-continuous replication.
Semi-continuous replication
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Section 2
Enzymology
of DNA Replication
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Enzymes and protein factors
protein Mr # function
Dna A protein 50,000 1 recognize origin
Dna B protein 300,000 6 open dsDNA
Dna C protein 29,000 1 assist Dna B binding
DNA pol Elongate the DNA strands
Dna G protein 60,000 1 synthesize RNA primer
SSB 75,600 4 single-strand binding
DNA topoisomerase 400,000 4 release supercoil constraint
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• The first DNA- dependent DNA polymerase (short for DNA-pol I) was discovered in 1958 by Arthur Kornberg who received Nobel Prize in physiology or medicine in 1959.
§2.1 DNA Polymerase
DNA-pol of prokaryotes
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• Later, DNA-pol II and DNA-pol III were identified in experiments using mutated E.coli cell line.
• All of them possess the following biological activity.
1. 53 polymerizing
2. exonuclease
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DNA-pol of E. coli
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DNA-pol I
• Mainly responsible for proofreading and filling the gaps, repairing DNA damage
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Klenow fragment
• small fragment (323 AA): having 5´→3´ exonuclease activity
• large fragment (604 AA): called Klenow fragment, having DNA polymerization and 3´→5´exonuclease activity
N end C end
caroid
DNA-pol Ⅰ
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DNA-pol II
• Temporary functional when DNA-pol I and DNA-pol III are not functional
• Still capable for doing synthesis on the damaged template
• Participating in DNA repairing
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DNA-pol III
• A heterodimer enzyme composed of ten different subunits
• Having the highest polymerization activity (105 nt/min)
• The true enzyme responsible for the elongation process
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Structure of DNA-pol III
α : has 5´→ 3´ polymerizing activity
ε : has 3´→ 5´ exonuclease activity and plays a key role to ensure the replication fidelity.
θ: maintain heterodimer structure
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DNA-pol of eukaryotes
DNA-pol : elongation DNA-pol III
DNA-pol : initiate replication and synthesize primers
DnaG, primase
DNA-pol : replication with low fidelity
DNA-pol : polymerization in mitochondria
DNA-pol : proofreading and filling gap
DNA-pol I
repairing
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§2.2 Primase
• Also called DnaG
• Primase is able to synthesize primers using free NTPs as the substrate and the ssDNA as the template.
• Primers are short RNA fragments of a several decades of nucleotides long.
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• Primers provide free 3´-OH groups to react with the -P atom of dNTP to form phosphoester bonds.
• Primase, DnaB, DnaC and an origin form a primosome complex at the initiation phase.
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§2.3 Helicase
• Also referred to as DnaB.
• It opens the double strand DNA with consuming ATP.
• The opening process with the assistance of DnaA and DnaC
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§2.4 SSB protein
• Stand for single strand DNA binding protein
• SSB protein maintains the DNA template in the single strand form in order to
• prevent the dsDNA formation;
• protect the vulnerable ssDNA from nucleases.
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§2.5 Topoisomerase
• Opening the dsDNA will create supercoil ahead of replication forks.
• The supercoil constraint needs to be released by topoisomerases.
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• The interconversion of topoisomers of dsDNA is catalyzed by a topoisomerase in a three-step process:
• Cleavage of one or both strands of DNA
• Passage of a segment of DNA through this break
• Resealing of the DNA break
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• Also called -protein in prokaryotes.
• It cuts a phosphoester bond on one DNA strand, rotates the broken DNA freely around the other strand to relax the constraint, and reseals the cut.
Topoisomerase I (topo I)
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• It is named gyrase in prokaryotes.
• It cuts phosphoester bonds on both strands of dsDNA, releases the supercoil constraint, and reforms the phosphoester bonds.
• It can change dsDNA into the negative supercoil state with consumption of ATP.
Topoisomerase II (topo II)
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3'
5'
5'
3'RNAase
POH
3'
5'
5'
3'
DNA polymerase
P
3'
5'
5'
3'
dNTP
DNA ligase
3'
5'
5'
3'
ATP
§2.6 DNA Ligase
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• Connect two adjacent ssDNA strands by joining the 3´-OH of one DNA strand to the 5´-P of another DNA strand.
• Sealing the nick in the process of replication, repairing, recombination, and splicing.
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§2.7 Replication Fidelity
• Replication based on the principle of base pairing is crucial to the high accuracy of the genetic information transfer.
• Enzymes use two mechanisms to ensure the replication fidelity.
– Proofreading and real-time correction
– Base selection
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• DNA-pol I has the function to correct the mismatched nucleotides.
• It identifies the mismatched nucleotide, removes it using the 3´- 5´ exonuclease activity, add a correct base, and continues the replication.
Proofreading and correction
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3´→5´ exonuclease activity excise mismatched
nuleotides
5´→3´ exonuclease activitycut primer or excise mutated segment
C T T C A G G A
G A A G T C C G G C G
5' 3'
3' 5'
Exonuclease functions
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Section 3
DNA Replication Process
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• Initiation: recognize the starting point, separate dsDNA, primer synthesis, …
• Elongation: add dNTPs to the existing strand, form phosphoester bonds, correct the mismatch bases, extending the DNA strand, …
• Termination: stop the replication
Sequential actions
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• The replication starts at a particular point called origin.
• The origin of E. coli, ori C, is at the location of 82.
• The structure of the origin is 248 bp long and AT-rich.
§3.1 Replication of prokaryotes
a. Initiation
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Genome of E. coli
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• Three 13 bp consensus sequences• Two pairs of anti-consensus repeats
Structure of ori C
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Formation of preprimosome
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• DnaA recognizes ori C.
• DnaB and DnaC join the DNA-DnaA complex, open the local AT-rich region, and move on the template downstream further to separate enough space.
• DnaA is replaced gradually.
• SSB protein binds the complex to stabilize ssDNA.
Formation of replication fork
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• Primase joins and forms a complex called primosome.
• Primase starts the synthesis of primers on the ssDNA template using NTP as the substrates in the 5´- 3´ direction at the expense of ATP.
• The short RNA fragments provide free 3´-OH groups for DNA elongation.
Primer synthesis
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• The supercoil constraints are generated ahead of the replication forks.
• Topoisomerase binds to the dsDNA region just before the replication forks to release the supercoil constraint.
• The negatively supercoiled DNA serves as a better template than the positively supercoiled DNA.
Releasing supercoil constraint
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Dna ADna B Dna C
DNA topomerase
5'3'
3'
5'
primase
Primosome complex
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• dNTPs are continuously connected to the primer or the nascent DNA chain by DNA-pol III.
• The core enzymes ( 、、 and ) catalyze the synthesis of leading and lagging strands, respectively.
• The nature of the chain elongation is the series formation of the phosphodiester bonds.
b. Elongation
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• The synthesis direction of the leading strand is the same as that of the replication fork.
• The synthesis direction of the latest Okazaki fragment is also the same as that of the replication fork.
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• Primers on Okazaki fragments are digested by RNase.
• The gaps are filled by DNA-pol I in the 5´→3´direction.
• The nick between the 5´end of one fragment and the 3´end of the next fragment is sealed by ligase.
Lagging strand synthesis
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3'
5'
5'
3'
RNAase
POH
3'
5'
5'
3'
DNA polymerase
P
3'
5'
5'
3'
dNTP
DNA ligase
3'
5'
5'
3'
ATP
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• The replication of E. coli is bidirectional from one origin, and the two replication forks must meet at one point called ter at 32.
• All the primers will be removed, and all the fragments will be connected by DNA-pol I and ligase.
c. Termination
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§3.2 Replication of Eukaryotes
• DNA replication is closely related with cell cycle.
• Multiple origins on one chromosome, and replications are activated in a sequential order rather than simultaneously.
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Cell cycle
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• The eukaryotic origins are shorter than that of E. coli.
• Requires DNA-pol (primase activity) and DNA-pol (polymerase activity and helicase activity).
• Needs topoisomerase and replication factors (RF) to assist.
Initiation
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• DNA replication and nucleosome assembling occur simultaneously.
• Overall replication speed is compatible with that of prokaryotes.
b. Elongation
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3'
5'
5'
3'
3'
5'
5'
3'
connection of discontinuous
3'
5'
5'
3'
3'
5'
5'
3'
segment
c. Termination
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• The terminal structure of eukaryotic DNA of chromosomes is called telomere.
• Telomere is composed of terminal DNA sequence and protein.
• The sequence of typical telomeres is rich in T and G.
• The telomere structure is crucial to keep the termini of chromosomes in the cell from becoming entangled and sticking to each other.
Telomere
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• The eukaryotic cells use telomerase to maintain the integrity of DNA telomere.
• The telomerase is composed of
telomerase RNA telomerase association protein telomerase reverse transcriptase
• It is able to synthesize DNA using RNA as the template.
Telomerase
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Inchworm model
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• Telomerase may play important roles is cancer cell biology and in cell aging.
Significance of Telomerase
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Section 4
Other Replication Modes
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§4.1 Reverse Transcription
• The genetic information carrier of some biological systems is ssRNA instead of dsDNA (such as ssRNA viruses).
• The information flow is from RNA to DNA, opposite to the normal process.
• This special replication mode is called reverse transcription.
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Viral infection of RNA virus
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Reverse transcription
Reverse transcription is a process in which ssRNA is used as the template to synthesize dsDNA.
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Process of Reverse transcription
• Synthesis of ssDNA complementary to ssRNA, forming a RNA-DNA hybrid.
• Hydrolysis of ssRNA in the RNA-DNA hybrid by RNase activity of reverse transcriptase, leaving ssDNA.
• Synthesis of the second ssDNA using the left ssDNA as the template, forming a DNA-DNA duplex.
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Reverse transcriptase
Reverse transcriptase is the enzyme for the reverse transcription. It has activity of three kinds of enzymes:
• RNA-dependent DNA polymerase
• RNase
• DNA-dependent DNA polymerase
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Significance of RT
• An important discovery in life science and molecular biology
• RNA plays a key role just like DNA in the genetic information transfer and gene expression process.
• RNA could be the molecule developed earlier than DNA in evolution.
• RT is the supplementary to the central dogma.
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Significance of RT
• This discovery enriches the understanding about the cancer-causing theory of viruses. (cancer genes in RT viruses, and HIV having RT function)
• Reverse transcriptase has become a extremely important tool in molecular biology to select the target genes.
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§4.3 D-loop Replication
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Section 5
DNA Damage and Repair
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Mutation is a change of nucleic acids in genomic DNA of an organism. The mutation could occur in the replication process as well as in other steps of life process.
§5.1 Mutation
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Consequences of mutation
• To create a diversity of the biological world; a natural evolution of biological systems
• To lead to the functional alternation of biomolecules, death of cells or tissues, and some diseases as well
• Changes of genotype, but no effect on phenotype
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§5.2 Causes of Mutation
DNA damage
UV radiation
viruses
carcinogensPhysical factors
evolution
infection
T
G
spontaneous mutation
Chemical modification
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N
N O
O
CH3
R
PN
N O
O
CH3
R
N
N O
O
CH3
R
P
N
N OR
UV
O
CH3
( T T )
)
Physical damage
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Mutation caused by chemicals
• Carcinogens can cause mutation.
• Carcinogens include: • Food additives and food preservative
s; spoiled food
• Pollutants: automobile emission; chemical wastes
• Chemicals: pesticides; alkyl derivatives; -NH2OH containing materials
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• Transition: the base alternation from purine to purine, or from pyrimidine to pyrimidine.
• Transversion: the base alternation between purine and pyrimidine, and vise versa.
Point mutation is referred to as the single nucleotide alternation.
a. Point mutation (mismatch)
§5.3 Types of Mutation
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Transition mutation
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HbS HbA
chains CAC CTC
mRNA GUG GAG
AA residue 6 in chain Val Glu
Hb mutation causing anemia
Single base mutation leads to one AA change, causing disease.
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b. Deletion and insertion
• Deletion: one or more nucleotides are
deleted from the DNA sequence.
• Insertion: one or more nucleotides are inserted into the DNA sequence.
Deletion and insertion can cause the reading frame shifted.
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Frame-shift mutation
Normal
5´… …GCA GUA CAU GUC … …
Ala Val His Val
Deletion C
5´… …GAG UAC AUG UC … …
Glu Tyr Met Ser
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c. Rearrangement
It is an exchange of large DNA fragments. It can be either reverse the direction or recombination between chromosomes.
1. Site-specific recombination
2. Homologous genetic recombination
3. DNA transposition
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• DNA repairing is a kind response made by cells after DNA damage occurs, which may resume their natural structures and normal biological functions.
• DNA repairing is a supplementary to the proofreading-correction mechanism in DNA replication.
§5.4 DNA Repairing
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N
N O
O
CH3
R
PN
N O
O
CH3
R
N
N O
O
CH3
R
P
N
N OR
UV
O
CH3
( T T )
)
Light repairing
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• One of the most important and effective repairing approach.
• UvrA and UvrB: recognize and bind the damaged region of DNA.
• UvrC: excise the damaged segment.
• DNA-pol Ⅰ: synthesize the DNA segment to fill the gap.
• DNA ligase: seal the nick.
Excision repairing
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• XP is an autosomal recessive genetic
disease. Patients will be suffered with hyper-sensitivity to UV which results in multiple skin cancers.
• The cause is due to the low enzymatic activity for the nucleotide excision-repairing process, particular thymine dimer.
Xeroderma pigmentosis (XP)
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Excision repairing
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Recombination repairing
• It is used for repairing when a large segment of DNA is damaged.
• Recombination protein RecA, RecB and RecC participate in this repairing.
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SOS repairing
• It is responsible for the situation that DNA is severely damaged and the replication is hard to continue.
• If workable, the cell could be survived, but may leave many errors.
• In E. coli, uvr gene and rec gene as well as Lex A protein constitute a regulatory network.