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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 bidirectional replication.
Semi-continuous replication
3
Leading strand
5
3
Fork shift
Fork
direction
move
direction Lagging strand
5
5
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.
DNA replication system
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
Section 2
Enzymology
of DNA Replication
The function of different DNA-pol of
prokaryotes
• Polymerase Ⅰ: Single peptide chain, the
function is proofreading and repairing .
• polymerase Ⅱ:Only has its activity without
DNA-pol Ⅰand Ⅲ.
• polymerase Ⅲ:It is the most effective
polymerase in synthesis of new strand DNA.
• All of DNA Polymerase possess the following
biological activity.
1. 53 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 : primase and extend the lagging
strand
DNA-pol : replication with low
fidelity
DNA-pol II
DNA-pol : polymerization in mitochondria
DNA-pol : elongation
DNA-pol III
DNA-pol : proofreading and
filling gap
DNA-pol I
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
Replication is a continuous process , to describe
it clearly, we separate it into three stage :
• 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.
§3.1 Replication of prokaryotes
a. Initiation
• The replication starts at a particular
point called origin.
• The origin of E. coli is 248 bp long
and AT-rich.
1
13
17
29
32
44
GATTNTTTATTT ··· GATCTNTTNTATT ··· GATCTCTTATTAG ···
3
5
5
3
Tandem Repeat Seq
Inverted Repeat Seq
···TGTGGATTA-‖-TTATACACA-‖-TTTGGATAA-‖-TTATCCACA
58
66
166
174
E.Coli oriC
201
209
237
245
Primosome complex
Dna A
Dna B Dna C
primase
3'
5'
3'
DNA topomerase
5'
SSB
Replication initiation related Pr. in procaryote
protein
Generic Name
Recognize ori C
DnaA (dnaA)
DnaB(dnaB)
Helicase
DnaC (dnaC)
DnaG (dnaG)
SSB
Topoisomerase
function
Unwinding
cooperate DnaB
Primase
synthesis primer
stabilize ssDNA
unwind supercoiled DNA
Releasing supercoil constraint
• 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.
b. Elongation
• dNTPs which is complementary to template are
continuously connected to the primer or the nascent
DNA chain by DNA-pol III.
• The nature of the chain elongation is the series
formation of the phosphodiester bonds.
(dNMP)n + dNTP
(dNMP)n+1 + PPi
DNA strand substrate
elongated
DNA strand
目录
Leading strand
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.
3'
5'
3'
3'
direction of unwinding
5'
5'
Semi-continuous replication
3'
5'
replication fork
3'
replication direction
3'
5'
5'
3'
5'
Okazaki fragment
3'
5'
leading strand
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
Proofreading and correction
• 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.
c. Termination
• 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.
Lagging strand synthesis
• 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.
Genome of E. coli
3'
5'
5'
3'
RNAase
3'
OH
5'
dNTP
3'
5'
DNA polymerase
P
ATP
3'
5'
P
5'
3'
5'
3'
DNA ligase
5'
3'
§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
Initiation
• 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.
b. Elongation
• DNA replication and nucleosome
assembling occur simultaneously.
• Overall replication speed is
compatible with that of prokaryotes.
c. Termination
3'
5'
5'
3'
3'
5'
5'
3'
3'
5'
connection of discontinuous
segment
5'
3'
3'
5'
5'
3'
Telomere
• 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.
Telomerase
• 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.
Inchworm model
Significance of Telomerase
• Telomerase may play important roles
in human aging and in cancer cell
biology .
aging
Cell division
Cell division
Target for
anti-cancer
telomerase
Immortalization
Section 4
Reverse Transcription
§4.1 Reverse Transcription
• Reverse transcription: is a process in
which 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 cancercausing 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
§5.1 Mutation
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.
§5.2 Causes of Mutation
UV radiation
Physical
factors
Chemical
modification
carcinogens
DNA
damage
infection
spontaneous
mutation
T
G
viruses
evolution
O
O
N
P
N
O
UV
O
N
R
R
CH3
N
N
O
CH3
P
CH3
O
R
N
O
N
CH3
O
)
R
N
(TT)
Mutation caused by chemicals
• Carcinogens can cause mutation.
• Carcinogens include:
• Food additives and food
preservatives; spoiled food
• Pollutants: automobile emission;
chemical wastes
• Chemicals: pesticides; alkyl
derivatives; -NH2OH containing
materials
§5.3 Types of Mutation
a. Point mutation (mismatch)
Point mutation is referred to as the
single nucleotide alternation.
5'
3'
3'
C T T C A G G A
G A A G T C C G G C G
5'
• Transition: the base alternation from
purine to purine, or from pyrimidine
to pyrimidine.
• Transversion: the base alternation
between purine and pyrimidine, and
vise versa.
Transition mutation
Hb mutation causing anemia
Single base mutation leads to one AA
change, causing disease.
HbS
HbA
 chains
CAC
CTC
 mRNA
GUG
GAG
AA residue 6 in  chain
Val
Glu
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
Chr 11
§5.4 DNA Repairing
• 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.
Light repairing
O
O
N
P
N
O
UV
O
N
R
R
CH3
N
N
O
CH3
P
CH3
O
R
N
O
N
CH3
O
)
R
N
(TT)
Excision repairing
Recognise and cleave
injured fragment by
endonuclease
Excision repairing
• 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.
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.