Characteristics of replication Semi-conservative replication Bidirectional replication...
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Transcript of Characteristics of replication Semi-conservative replication Bidirectional replication...
Characteristics of replication
Semi-conservative replication
Bidirectional replication
Semi-continuous replication
§1.1 Semi-Conservative Replication
• Conception: The original double-stranded DNA opens up and both strands serve as template for the synthesis of new DNA, The products of the reaction are two daughter double stranded DNA molecules. each of them has one original template strand and one newly synthesized strand DNA.
§1.2 Bidirectional Replication• Bi-directional: replication is started in a
single origin C site , extending in two direction , It process is called bi-directional replication.
Semi-continuous replication
3
5
3
5Fork move direction
3´
5´
3´
3´
5´ Fork shift dir
ection
5
Leading strand
Lagging strand
Synthesis direction of leading strand accord with the replication fork shift direction, and synthesis direction of lagging strand is against the replication fork shift direction.
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
Section 2
Enzymology
of DNA Replication
The function of different DNA-pol of prokaryotes
• Polymerase : Single peptide chain, the Ⅰfunction is proofreading and repairing .
• polymerase :Only has its activity Ⅱ without DNA-pol and .Ⅰ Ⅲ
• polymerase :It is the Ⅲ most effective polymerase in synthesis of new strand DNA.
• All of DNA Polymerase possess the following biological activity.
1. 53 polymerizing(for replication)
• Function: Recognizes the deoxynucleotide on the DNA template and then adds a complementary dNTP to the 3’-OH of the primer ,creating a 3’,5’phosphodiester bond on the daughter strand.
2. exonuclease (for proofreading)• Function: Check the base pair between parent
and daughter strand starting from the end of DNA,and remove mismatched deoxynucleotide from daughter strand.
DNA-pol of eukaryotes
DNA-pol : elongation DNA-pol III
DNA-pol : primase and extend the lagging strand
DNA-pol : replication with low fidelity
DNA-pol : polymerization in mitochondria
DNA-pol : proofreading and filling gap
DNA-pol I
DNA-pol II
The initiation phase of DNA replication
§2.3 Helicase
• Unwinding of double helix DNA is required before replication, because the template must paired with its complement dNTP. Helicase can use the ATP to unwind double helix DNA forming a replication fork.
§2.4 SSB protein
SSB(single-stranded DNA binding protein):
When DNA was unwinded into two single strand by helicase, it intend to form double-stranded DNA again.SSB binds to single strand DNA and keep the single state of DNA in replication.
§2.5 Topoisomerase• During unwinding double helix DNA, the
downstream double helix DNA over wrapped.
• The superhelix template needs to be released by topoisomerases.
§2.6 DNA Ligase
• During replication , the synthesis of new strand may be discontinuous , to form many okazaki fragment . the DNA ligase can link two adjacent fragment by 3’-5’phosphodiester bond ,form the integrated daughter strand.
Section 3
DNA Replication Process
• Initiation: recognize the origine point, separate dsDNA, primer synthesis, …
• Elongation: add dNTPs to the existing strand, form phosphodiester bonds, correct the mismatch bases, extending the DNA strand, …
• Termination: stop the replication.
Replication is a continuous process , to describe it clearly, we separate it into three stage :
• The replication starts at a particular point called origin.
• The origin of E. coli is 248 bp long and AT-rich.
§3.1 Replication of prokaryotes
a. Initiation
GATTNTTTATTT ··· GATCTNTTNTATT ··· GATCTCTTATTAG ···
53
E.Coli oriC
1 13 17 29 32 44
···TGTGGATTA-‖-TTATACACA-‖-TTTGGATAA-‖-TTATCCACA
58 66 166 174 201 209 237 245
Tandem Repeat Seq Inverted Repeat Seq
53
Dna ADna B Dna C
DNA topomerase
5'3'
3'
5'
primase
Primosome complex
SSB
Primase synthesis primerDnaG (dnaG)
cooperate DnaBDnaC (dnaC)
UnwindingHelicaseDnaB(dnaB)
Recognize ori CDnaA (dnaA)
protein Generic Name function
Replication initiation related Pr. in procaryote
SSB stabilize ssDNA
Topoisomerase unwind supercoiled DNA
• 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
目 录
• dNTPs which is complementary to template are cont
inuously connected to the primer or the nascent DN
A chain by DNA-pol III.
• The nature of the chain elongation is the series form
ation of the phosphodiester bonds.
b. Elongation
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
Semi-continuous replication
3'
5'
5'3'
replication direction
Okazaki fragment
3'
5'
leading strand
3'
5'
3'
5'replication fork
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
• 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
• 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
• 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
Genome of E. coli
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
§3.2 Replication of Eukaryotes
• DNA replication is closely related with cell cycle.
• Multiple origins on one chromosome, and replications are activated simultaneously.
Cell cycle
• The eukaryotic origins are shorter than that of E. coli.
• Requires DNA-pol (primase activity) and DNA-pol (polymerase activity).
• Needs topoisomerase and replication factors (RF) to assist.
Initiation
• DNA replication and nucleosome assembling occur simultaneously.
• Overall replication speed is compatible with that of prokaryotes.
b. Elongation
3'
5'
5'
3'
3'
5'
5'
3'
connection of discontinuous
3'
5'
5'
3'
3'
5'
5'
3'
segment
c. Termination
• 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 TTAGGG repeat sequence.
• The telomere structure is crucial to keep the termini of chromosomes in the cell from becoming entangled and sticking to each other.
Telomere
• 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
Inchworm model
• Telomerase may play important roles in human aging and in cancer cell biology .
Significance of Telomerase
Cell Cell divisiondivision
Cell divisionCell division
aging
telomerasetelomeraseTarget for Target for anti-canceranti-cancer
ImmortalizationImmortalization
Section 4
Reverse Transcription
§4.1 Reverse Transcription• Reverse transcription: is a process in whic
h genetic information is transmitted from RNA to DNA . It’s phenomenon often occure in the process that eukaryote cell be infected by RNA virus.
• In these RNA virus,the genetic information carrier is ssRNA instead of dsDNA (such as ssRNA viruses,HIV virus or tumour virus).
§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.
Viral infection of RNA virus
During RNA virus infect Host cell,the genetic information must be transmitted from ssRNA to ds DNA ,and integrated into the chromosome of Host cell.Then replicate and transcribe with the DNA from Host cell.
Reverse transcription
Reverse transcription is a process in which ssRNA is used as the template to synthesize dsDNA.
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.
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
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.
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.
Section 5
DNA Damage and Repair
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
§5.2 Causes of Mutation
DNA damage
UV radiation
viruses
carcinogensPhysical factors
evolution
infection
T
G
spontaneous mutation
Chemical modification
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 )
)
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
Point mutation is referred to as the single nucleotide alternation.
a. Point mutation (mismatch)
§5.3 Types of Mutation
C T T C A G G A
G A A G T C C G G C G
5' 3'
3' 5'
• Transition: the base alternation from
purine to purine, or from pyrimidine t
o pyrimidine.
• Transversion: the base alternation be
tween purine and pyrimidine, and vis
e versa.
Transition mutation
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.
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.
Frame-shift mutation
Normal
5´… …GCA GUA CAU GUC A… …
Ala Val His Val
Deletion C
5´… …GAG UAC AUG UCA … …
Glu Tyr Met Ser
c. Rearrangement
It is the exchang or transfer of genetic information between homologous chromosome ,resulting in the formation of new characteristics not found in either parental DNA.
Gene Rrarrangement lead to mediterranean anemia
Hb β gene family on Ch
r 11
• 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
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
Excision repairing
Recognise and cleave injured fragment by endonuclease
• 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
Recombination repairing
• It is used for repairing when a large segment of DNA is damaged.
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.