DNA RNA protein transcriptiontranslationreplication reverse
transcription Central dogma
Slide 4
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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Slide 6
Lecture 2 DNA Replication DNA Biosynthesis
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Section 1 General Concepts of DNA Replication
Slide 8
Double helix structure of DNA It has not escaped our notice
that the specific pairing we have postulated immediately suggests a
possible copying mechanism for the genetic material.Watson &
Crick
Slide 9
Characteristics of replication Semi-conservative replication
Bidirectional replication Semi-continuous replication
Slide 10
1.1 Semi-Conservative Replication Meselson and Stahl
(1958)
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Semiconservative replication Definition: Half of the parental
DNA molecule is conserved in each new double helix, paired with a
newly synthesized complementary strand. Significance: The genetic
information is ensured to be transferred from one generation to the
next generation with a high fidelity. ATTGCATTGC TAACGTAACG
ATTGCATTGC TAACGTAACG ATTGCATTGC TAACGTAACG Parent molecule
Daughter molecule
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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. Origin
Examination of T7 DNA replication using electron microscopy
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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 synthesis
continue.
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Replication of prokaryotes The replication process starts from
the origin, and proceeds in two opposite directions. It is named
replication.
Slide 15
Replication of eukaryotes Chromosomes of eukaryotes have
multiple origins.
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The DNA strands are antiparallel. At a replication fork, both
strands of parental DNA serve as templates for the synthesis of new
DNA; All known DNA polymerases synthesize DNA in the 5 3 direction
but not in 3 5 direction. ? 1.3 Semi-continuous Replication
Slide 17
This dilemma was resolved by Reiji Okazaki ( in the 1960s), who
found that a significant proportion of newly synthesized DNA exists
as small fragments; These units of about a thousand nucleotides are
called Okazaki fragments; They are 1000 2000nt long for prokaryotes
and 100- 150nt long for eukaryotes. Reiji Okazaki and his wife
Tsuneko Okazaki
Slide 18
The leading strand :the strand synthesized continuously; The
lagging strand :the strand formed from Okazaki fragments; The
semi-continuous replication: 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
Slide 19
Section 2 Enzymology of DNA Replication Large team of enzymes
coordinates replication Let s meet the team
Slide 20
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
Slide 21
Daughter strand synthesis Chemical formulation: The nature of
DNA replication is a series of 3,5phosphodiester bond formation
catalyzed by a group of enzymes.
Slide 22
Phosphodiester bond formation
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(dNMP) n + dNTP (dNMP) n+1 + PPi Where s the ENERGY for the
bonding! energy We come with our own energy!
Slide 24
Enzymes and protein factors proteinMrMr #function Dna A
protein50,0001recognize origin Dna B protein300,0006open dsDNA Dna
C protein29,0001assist Dna B binding DNA polElongate the DNA
strands Dna G protein60,0001synthesize RNA primer
SSB75,6004single-strand binding DNA topoisomerase400,0004release
supercoil constraint
Slide 25
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
Slide 26
Arthur Kornberg (left) with his son, Roger, after Roger
received the 2006 Nobel Prize in Chemistry. Kornberg liked to refer
to his scientific career as a "love affair with enzymes."
Slide 27
Later, DNA-pol II and DNA-pol III were identified in
experiments using mutated E.coli cell line. DNA-pol I possess the
following biological activity. 1. 5 3 polymerizing 2. The 3` to 5`
exonuclease activity 3. The 5` to 3` exonuclease activity Why does
a DNA polymerase also need two exonuclease activities?
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5 3 polymerizing 3 5 3 5
Slide 29
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 activity cut primer or excise mutated segment
Exonuclease functions
Slide 31
DNA-pol of E. coli
Slide 32
DNA-pol I Function mainly responsible for proofreading and
filling the gaps, repairing DNA damage
Slide 33
Klenow fragment Klenow fragment: large fragment (604 AA) of DNA
pol I, having DNA polymerization and 3 5exonuclease activities, and
is widely used in molecular biology.
Slide 34
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 (10 5 nt/min)
The true enzyme responsible for the elongation process
Slide 36
Structure of DNA-pol III has 5 3 polymerizing activity has 3 5
exonuclease activity and plays a key role to ensure the replication
fidelity. : maintain heterodimer structure
Slide 37
DNA Polymerase III- does the bulk of copying DNA in Replication
2 subunit: sliding clamp
Slide 38
2.2 Primase Also called DnaG Primase (a specific RNA
polymerase) : synthesize primers using free NTPs as the substrate
and the ssDNA as the template. Primers: short RNA fragments (5-50
nucleotides).
Slide 39
2.3 Helicase Also referred to as DnaB. It opens the double
strand DNA with consuming ATP. (Zip opener) The opening process
with the assistance of DnaA and DnaC Dna B Dna C
Slide 40
2.4 SSB protein( single strand DNA binding protein) maintains
the DNA template in the single strand form in order to prevent the
dsDNA formation; protect the ssDNA degradation by nucleases.
Slide 41
2.5 Topoisomerase Opening the dsDNA will create supercoil ahead
of replication forks, the supercoil constraint needs to be released
by topoisomerases (type I and II).
Slide 42
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
Slide 43
It is named gyrase in prokaryotes. It cuts phosphoester bonds
on both strands of dsDNA, releases the supercoil constraint, and
reforms the phosphoester bonds. Topoisomerase II Antibiotics:
ciprofloxacin, novobiocin and nalidixic acid, inhibit the bacterial
gyrase. Anticancer agents: adriamycin, etoposide, and doxorubicin,
inhibit human topoisomerase.
Slide 44
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.
2.6 DNA Ligase HO 5 33 33 55 DNA Ligase ATP NAD + AMP 55 33 55
33
Slide 45
Section 3 DNA Replication Process
Slide 46
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
Slide 47
The replication starts at a particular point called origin. 3.1
Replication of prokaryotes a. Initiation Genome of E. coli
Slide 48
DNA sequences at the Bacterial origin of Replication The
structure of the origin is 248 bp long and AT-rich.
Slide 49
DnaA recognizes origin. DnaB(helicase) and DnaC join the DNA-
DnaA complex, open the local AT-rich region, and move on the
template downstream further to separate enough space. SSB protein
binds the complex to stabilize ssDNA. Formation of replication
fork
Slide 50
Primase joins and starts the synthesis of RNA primers.
Primasome: protein complex responsible for creating RNA primers on
ssDNA during DNA replication. Topoisomerase binds to the dsDNA
region just before the replication forks to release the supercoil
constraint. Primer synthesis
Slide 51
3 5 3 5 primer 3' HO 5' primase The short RNA fragments provide
free 3-OH groups for DNA elongation.
Slide 52
dNTPs 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. b. Elongation
Slide 53
Slide 54
Primers on Okazaki fragments are digested by RNase. The gaps
are filled by DNA-pol I in the 5 3direction. The nick between the
5end of one fragment and the 3end of the next fragment is sealed by
DNA ligase. Lagging strand synthesis RNase DNA-pol I
Slide 55
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.
flash
Slide 56
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 ori ter 82 32
movie
Slide 57
Replication of prokaryotes The replication process starts from
the origin, and proceeds in two opposite directions. It is named
replication.
Slide 58
Replication Fidelity Replication based on the principle of base
pairing is crucial to the high accuracy of the genetic information
transfer. Enzymes use three mechanisms to ensure the replication
fidelity.
Slide 59
110 -5 110 -5 110 -2 110 -9
Slide 60
3.2 Replication of Eukaryotes DNA replication is closely
related with cell cycle: S- phase. Multiple origins on one
chromosome. Cell cycle
Slide 61
DNA-pol of eukaryotes DNA-pol : elongationDNA-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
Slide 62
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
Slide 63
DNA replication and nucleosome assembling occur simultaneously.
Overall replication speed is compatible with that of prokaryotes.
b. Elongation 3 5 5 3 Leading strand 3 5 3 5 Lagging strand primer
nucleosome
Slide 64
c. Termination
Slide 65
The End Replication Problem: Telomeres shorten with each S
phase Ori 3' 5' 3' 5' 3' 5'
Slide 66
Telomere: the terminal structure of eukaryotic DNA of
chromosomes. composed of terminal DNA sequence and protein.
Function: keep the termini of chromosomes in the cell from becoming
entangled and sticking to each other. Telomere shoelace Repetitive
DNA sequence (TTAGGG in vertebrates) Form a 'capped' end
structure
Slide 67
The Nobel Prize in Physiology or Medicine 2009 Elizabeth
BlackburnCarol GreiderJack Szostak "for the discovery of how
chromosomes are protected by telomeres and the enzyme
telomerase"
Slide 68
Telomerase: the enzyme that essentially builds new telomeres,
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
Slide 69
Inchworm model inchworm
Slide 70
Slide 71
Telomerase is highly active in the embryo, and after birth it
is active in the reproductive and stem cells. Telomerase may play
important roles in cell aging and cancer cell biology. Significance
of Telomerase
Slide 72
In most somatic tissues, telomerase is expressed at very low
levels or not at all -- as cells divide, telomeres shorten
Telomerase and Senescence Short telomeres signal cells to senesce
(stop dividing) cellular clock
Slide 73
Telomerase and Cancer Strong evidence to suggest that the
absence of senesence in cancer cells is linked to the activation of
the telomerase. Telomerase is an attractive target for cancer
chemotherapy.
Slide 74
SUMMARY Telomeres are essential for chromosome stability
Telomere shortening occurs owing to the biochemistry of DNA
replication Short telomeres cause replicative senescence Telomerase
prevents telomere shortening and replicative senescence
Slide 75
Section 4 Reverse Transcription
Slide 76
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.
Slide 77
Viral infection of RNA virus
Slide 78
Reverse transcription Reverse transcription is a process in
which ssRNA is used as the template to synthesize dsDNA. Synthesis
of ssDNA complementary to ssRNA, cDNA, forming a RNA-DNA hybrid.
Hydrolysis of ssRNA: RNase activity of reverse transcriptase,
leaving ssDNA. Synthesis of the second ssDNA, forming a DNA-DNA
duplex.
Slide 79
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
Slide 80
David Baltimore In 1970 Discover RNA-dependant DNA polymerase
which later known as reverse transcriptase. 1975 Nobel Prize in
Physiology or Medicine Howard M. Temin
Slide 81
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.
Slide 82
Section 5 DNA Damage and Repair
Slide 83
Definition: 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. 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 5.1 Mutation
Slide 84
5.2 Causes of Mutation
Slide 85
Physical damage
Slide 86
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; nitrous acid(HNO 2 )
Slide 87
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
Slide 88
Nitrous acid (HNO 2 ): react with base that contain amino
groups, deaminates C to produce U, resulting in GC AU Nitrous acid
formed by digestion of nitrites (preservatives) in foods.
Slide 89
Consequences of point mutations Silent mutation: The code
containing the changed base may code for the same amino acid. UCA,
UCU, all code for serine. Missense mutation: the changed base may
code for a different amino acid. UCA for serine, ACA for threonine.
Nonsense mutation: the codon with the altered base may become a
termination codon. UCA for serine, UAA for stop codon.
Slide 90
HbSHbA chains CACCACCTCCTC mRNA GUGGUGGAGGAG AA residue 6 in
chain ValGlu Hb mutation causing anemia Single base mutation leads
to one AA change, causing disease.
Slide 91
Slide 92
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.
Slide 93
Frame-shift mutation Normal 5 GCA GUA CAU GUC Ala Val His Val
Deletion C 5 GAG UAC AUG UC Glu Tyr Met Ser
Slide 94
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
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 : synthesize the DNA segment to
fill the gap. DNA ligase: seal the nick. Excision repairing UvrA
UvrB UvrC OH P DNA-pol OH P DNA ligase NAD +
Slide 97
XP is an 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
pigmentosum (XP) The most obvious, and often important part of
treatment is avoiding exposure to sunlight.
Slide 98
It is used for repairing when a large segment of DNA is
damaged. Recombination protein RecA, RecB and RecC participate in
this repairing. Recombination repairing
Slide 99
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.
Slide 100
Points I. General characteristics Semi-conservative; Specific
origins; Bidirectional; Semidiscontinuous replication II. Bacterial
Replication A. Polymerization 1. template, primer, dNTP, proceed in
5` to 3` direction 2. Pol I, Pol II, Pol III 3. other replication
proteins at the replication fork SSB, helicase, topoisomerase B.
Semidiscontinuous replication: leading strand and lagging strand
synthesis 1. RNA primer synthesized by the primases 2.
polymerization by Pol III 3. completion by Pol I and ligase 4.
Okazaki fragment
Slide 101
Points (continue) . Eukaryotic Replication S phase; Telomere
and Telomerase . Reverse transcription Definition; Significance .
Mutation, DNA damage and repair Point mutation; insertion and
deletion, Frameshift mutations Physical and chemical damage;
photoreactivation repair; excision repair Xeroderma pigmentosum
(XP)
Slide 102
Key terms Semiconservative replication Replication fork
Semidiscontinuous replication DNA polymerase, DNA ligase Template,
primer, Okazaki fragments Leading strand, lagging strand Reverse
transcription
Slide 103
Concepts and terms to understand: The difference between a
template and a primer? The difference between primase and
polymerase? Why are single-stranded binding (SSB) proteins
required? How does synthesis differ on leading strand and lagging
strand?