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OBJECTIVES
Review the chemistry of the genetic information
carrier.
Enumerate the requirements in the synthesis of DNA.
Discuss the role of proteins in DNA synthesis.
Discuss the chemical reactions involved in the
synthesis of DNA.
Compare and contrast the synthesis of DNA in
eukaryotes and prokaryotes.
Relate the role of gene expression to the practice of
Medicine.GENE EXPRESSION 1
MAVillamo r, MD,MHPEdDepartment of B iochemistry
Januar 2012
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GENE EXPRESSION
Components of the information unitof living organisms
Central dogma of molecular biology
Metabolism of chromosomal DNA
REPLICATION
REPAIR
RECOMBINATIONGENE EXPRESSION 1
MAVillamo r, MD,MHPEdDepartment of B iochemistry
Januar 2012
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GENE
DNA
BIOLOGICPRODUCTS
TEMPLATE
POLYPEPTIDE
CHAINs
RNAs
to
synthesize
including
DOUBLEHELIX
dNTPs
base-pair
REGULATORY
sequences
CODINGsequences
organized into
contains
Initiation Termination
GENE EXPRESSION
to denote
of
serves
as
composed of
GENE MOLECULAR NATURE
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DEOXYRIBONUCLEIC ACID
(DNA)
Repository of geneticinformation
Encode the primary structures
Cellular RNAs
ProteinsRIBONUCLEIC ACID (RNA)
GENE EXPRESSION 1
MAVillamo r, MD,MHPEdDepartment of B iochemistry
Januar 2012
NUCLEIC ACIDS
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ORGANISM Total DNA Number ofChromosomes Number ofGenes
E.coli 4,639,221 1 4,405
Yeast 12,068,000 16 6,200
Fruit fly 180,000,000 18 13,600
Mouse 2,500,000,000 40 30,000-35,000
Human 3,200,000,000 46 30,000-35,000
GENE EXPRESSION 1
MAVillamo r, MD,MHPEdDepartment of B iochemistry
Januar 2012
CHROMOSOMAL COMPOSITION
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GENE EXPRESSION 1
MAVillamo r, MD,MHPEdDepartment of B iochemistry
Januar 2012
DNA CHEMISTRY
PO4
H2C
PO4
O
1
5
3
TN
O
N
O
PO4
3
CH2
1 5
O
A NN
N
PO4
H
H
H
H
H
5 End
5 End3 End
3 End
H2C
O
1
5
3
CN
N
N
O
PO4
3
CH2
1 5
O
GNN
N
O
PO4
4
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CHEMISTRY: DNA
GENE EXPRESSION 1
MAVillamo r, MD,MHPEdDepartment of B iochemistry
Januar 2012
Right - handed helixBase per turn
9.7
Pitch per turn
33.2 A
Longer and thinner
Rise per bp
3.5 A
Right - handed helix
Base per turn
10.7
Pitch per turn
24.6 A
Short and broad
Rise per bp
2.6 A
Left - handed helix
Base per turn
12
Pitch per turn
45.6 A
Elongated and thin
Rise per bp
3.7 A (crystal)
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EUKARYOTIC CHROMATIN
GENE EXPRESSION 1
MAVillamo r, MD,MHPEdDepartment of B iochemistry
Januar 2012
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CHEMISTRY: MITOCHONDRIAL DNA
GENE EXPRESSION 1
MAVillamo r, MD,MHPEdDepartment of B iochemistry
Januar 2012
Circular double-stranded DNA Structural genes: 13 proteins of ETC
Encodes 22 mt tRNA molecules
COMPLEX SUBUNITSSUBUNITS
ENCODED
INADH Ubiquinone oxidoreductase >40 7
IISuccinate dehydrogenase 4 0
IIIUbiquinol-cytochrome c oxidoreductase 11 1
IVCytochrome c oxidase 13 3
VATP synthase 12 2
DEVLIN ( 2008). Textbook of Biochemistry
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DNARNA
TRANSCRIPTION
Polypeptide Chain
TRANSLATION
Post-translationalModification
Functional Protein
REPLICATION
REVERSE
TRANSCRIPTION
GENE EXPRESSION 1
MAVillamo r, MD,MHPEdDepartment of B iochemistry
Januar 2012
CENTRAL DOGMA
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GENE EXPRESSION 1
MAVillamo r, MD,MHPEdDepartment of B iochemistry
Januar 2012
DNA SYNTHESIS
Prokaryotic
replication
Eukaryotic
replicationMECHANISM Semiconservative
ORIGINSingle origin replication
(oriC)
Multiple origins of replication(ARS)
PRIMER SYNTHESIS Primase DNA polymerase subunits
PROCESSIVE ENZYME DNA polymerase IIIDNA polymerases
and
RNA REMOVAL DNA polymerase IDNA polymerase
DNA Nucleoid Chromatin
Circular DNA Linear DNA
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Template dependentBegins at an origin and proceeds
bidirectionally
Proceeds in a5 to 3 directionSemidiscontinuous
Semiconservative
GENE EXPRESSION 1
MAVillamo r, MD,MHPEdDepartment of B iochemistry
Januar 2012
FUNDAMENTAL RULES - Replication
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alexandria.healthlibrary.ca
GENE EXPRESSION 1
MAVillamo r, MD,MHPEdDepartment of B iochemistry
Januar 2012
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* Origin of replication
* Denaturation of dsDNA
* Formation of the replication fork
*
Synthesis of RNA primerELONGATION
TERMINATION
* Polymerization of daughter strands
* Reannealing
* Reconstitution of chromatinstructure
INITIATION
GENE EXPRESSION 1
MAVillamo r, MD,MHPEdDepartment of B iochemistry
Januar 2011
DNA SYNTHESIS:PHASES
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DNA SYNTHESIS: INITIATION
5 End 3 End
5 End3 End
5 End 3 End
5 End3 End
5 End 3 End
5 End3 End
OriC
GENE EXPRESSION 1
MAVillamo r, MD,MHPEdDepartment of B iochemistry
Januar 2012
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GENE EXPRESSION 1
MAVillamo r, MD,MHPEdDepartment of B iochemistry
Januar 2011
DNA SYNTHESIS: PROTEIN REQUIREMENTS
* Deoxynucleotide polymerization
* Processive unwinding of DNA
* Relieve torsional strain
* Initiates synthesis of RNA primer
*Prevent premature re-annealing ofdsDNA
DNA POLYMERASES
HELICASES
TOPOISOMERASES
DNA PRIMASE
SINGLE-STRAND BP
DNA LIGASE* Seals single strand nick
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GENE EXPRESSION 1
MAVillamo r, MD,MHPEdDepartment of B iochemistry
Januar 2011
TOPOISOMERASE
Topoisomerase I
Tyr
5 End 3 End
5 End3 End
PO4
PO4
PO4
PO4
PO4
PO4
PO4
PO4
Introduce a transient breakTransesterification
O O O SO S
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INITIATION: TOPOISOMERASE I
GENE EXPRESSION 1
MAVillamo r, MD,MHPEdDepartment of B iochemistry
Januar 2012
INITIATION TOPOISOMERASE II
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INITIATION: TOPOISOMERASE II
GENE EXPRESSION 1
MAVillamo r, MD,MHPEdDepartment of B iochemistry
Januar 2012
Novobiocin
Nalidixic acid
DNA SYNTHESIS INITIATION
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* SEPARATION OF DNA STRANDS*
Helicases* bind to a single strand
* move along the strand in a fixed directionrequiring ATP
* Single
stranded DNA binding proteins(SSBs)
* reduce potential secondary structure
* keep the DNA strands apart
* align the template strand for rapid DNAsynthesis
DNA SYNTHESIS: INITIATION
GENE EXPRESSION 1
MAVillamo r, MD,MHPEdDepartment of B iochemistry
Januar 2012
INITIATION HELICASE
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INITIATION: HELICASE
GENE EXPRESSION 1
MAVillamo r, MD,MHPEdDepartment of B iochemistry
Januar 2012
INITIATION HELICASE
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INITIATION: HELICASE
GENE EXPRESSION 1
MAVillamo r, MD,MHPEdDepartment of B iochemistry
Januar 2012
DNA REPLICATION REACTIONS
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PRIMING
Nucleophilic attack ofa
phosphate of dNTP
Primer dependent
RNA with 10
60 nucleotidesProvides a free 3 OHfor addition of
dNTP
Synthesized by PrimaseDNA dependent RNA polymerase
DNA REPLICATION: REACTIONS
GENE EXPRESSION 1
MAVillamo r, MD,MHPEdDepartment of B iochemistry
Januar 2012
DNA SYNTHESIS PRIMING
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3
CH21
5
O
ANN
N
PO4
H
H
H
H
H
5 End
3 End
PO4
H2C
PO4
O
1
5
3
TN
O
N
O
5 End
3 End
H2C
O1
5
3
CN
N
N
O
PO4
PO4 PO4
3
CH2
1 5
O
GNN
N
O
RNA PRIMER
GENE EXPRESSION 1
MAVillamo r, MD,MHPEdDepartment of B iochemistry
Januar 2011
DNA SYNTHESIS: PRIMING
PRIMER SYNTHESIS
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GENE EXPRESSION 1
MAVillamo r, MD,MHPEdDepartment of B iochemistry
Januar 2011
PRIMER SYNTHESIS
* Short RNA stretches of 810
nucleotides
* Provides the free 3 OH
terminus for NTP addition
DNA SYNTHESIS INITIATION
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GENE EXPRESSION 1
MAVillamo r, MD,MHPEdDepartment of B iochemistry
Januar 2011
DNA SYNTHESIS: INITIATION
Initiator protein
(DnaA)
Origin of replication
HELICASE(DnaB)
5
53
3
SSBPRIMASE
DNA
POLYMERASE
DnaC
INITIATION f REPLICATION E li
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* OriC recognitionDnaA Protein
* DNA unwindingDnaB Protein
* Binding of DnaB to originDnaC Protein
* Primer synthesisDnaG Protein
* Binds to ssDNASSB
* Relieves torsion during unwindingDNA gyrase
* Methylation of GATC at OriCDam methylase
* DNAbinding proteins that stimulatesinitiation
FIS/ IHF
* Histone like protein that stimulates initiationHU
GENE EXPRESSION 1
MAVillamo r, MD,MHPEdDepartment of B iochemistry
Januar 2011
INITIATION of REPLICATION E.coli
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DNA SYNTHESIS:ELONGATION
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* DNA SYNTHESIS
DNA polymerase Chain Elongation
Phosphodiester bond formation
Addition of nucleotides to the 3- OHends Processivity
Proofreading
* ELONGATION from the 5to 3end Leading strandcontinuous elongation
Lagging stranddiscontinuous elongation Okazaki fragment (130200 NTPs)
Retrograde strand
GENE EXPRESSION 1
MAVillamo r, MD,MHPEdDepartment of B iochemistry
Januar 2011
DNA SYNTHESIS:ELONGATION
DNA SYNTHESIS: ELONGATION
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GENE EXPRESSION 1
MAVillamo r, MD,MHPEdDepartment of B iochemistry
Januar 2011
DNA SYNTHESIS: ELONGATION
5
3
3
5
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ELONGATION: DNA POLYMERASE
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ELONGATION: DNA POLYMERASE
GENE EXPRESSION 1
MAVillamo r, MD,MHPEdDepartment of B iochemistry
Januar 2012
DNA POLYMERASE
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DNA POLYMERASE
DnaB
Helicase
t
t
t
b
g
bClamp
Core(aeq
)Core(aeq)
GENE EXPRESSION 1
MAVillamo r, MD,MHPEdDepartment of B iochemistry
Januar 2012
DNA POLYMERASE III: SUBUNITS
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DNA POLYMERASE III: SUBUNITS
GENE EXPRESSION 1
MAVillamo r, MD,MHPEdDepartment of B iochemistry
Januar 2012
SUBUNIT GENE FUNCTION
a
polC Polymerization activity
e dnaQ 3 to 5 proofreading exonuclease
q
holE Stabilization of esubunit
t
dnaX Stable template binding
g dnaX* Clamp loader
d
holA Clamp loader
d holB Clamp loader
c holC Interaction with SSBy
holD Interaction with gand c
b
dnaN DNA clamp for processivity
DNA SYNTHESIS: ELONGATION
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DNA SYNTHESIS: ELONGATION
5
53
3
REPLICATION FORK MOVEMENT
LAGGING STRAND
Discontinuous strand
Retrograde strand
Okazaki fragments
- 130200 bp
LEADING STRAND
Continuous strand
Processive strand
Anteriograde
GENE EXPRESSION 1
MAVillamo r, MD,MHPEdDepartment of B iochemistry
Januar 2012
DNA SYNTHESIS: ELONGATION
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GENE EXPRESSION 1
MAVillamo r, MD,MHPEdDepartment of B iochemistry
Januar 2011
DNA SYNTHESIS: ELONGATION
DNA SYNTHESIS: ELONGATION
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DNA SYNTHESIS: ELONGATION
GENE EXPRESSION 1
MAVillamo r, MD,MHPEd
Department of B iochemistry
Januar 2012
Model of T7 DNA replication.
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DNA SYNTHESIS: TERMINATION E coli
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DNA SYNTHESIS: TERMINATION E.coli
Replication forks meet at a terminus region
TerFunctions as binding site for Tus
TerTus complex can arrest one replication fork
OriC
GENE EXPRESSION 1
MAVillamo r, MD,MHPEd
Department of B iochemistryJanuary 2012
TERMINATION: GAP FILLING
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GAPS
Formed after removal of a primer
At least one nucleotide is missing
Filled with dNTPs by DNA polymerase
TERMINATION: GAP FILLING
GENE EXPRESSION 1
MAVillamo r, MD,MHPEd
Department of B iochemistry
Januar 2012
LIGATION
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NICK
Interruption in the phosphodiester backbone
LIGATION
Nick sealing
Phosphodiester bond formation
DNA ligase
Requires energyATP or NAD
+
LIGATION
GENE EXPRESSION 1
MAVillamo r, MD,MHPEd
Department of B iochemistry
Januar 2012
LIGATION
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LIGATION
GENE EXPRESSION 1
MAVillamo r, MD,MHPEd
Department of B iochemistry
Januar 2012
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DNA SYNTHESIS: PROTEINS
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FUNCTION E.Coli Eukaryote
Recognition of origin of replication DnaA ORC Proteins
Relieves positive supercoils Gyrase Toposiomerase I/II
Unwinds parental duplex DnaB Mcm
Loads helicase to DNA DnaC Cdc6, Cdt1
Maintains DNA in single-stranded state SSB RPA
Load clamp into DNAg
- complex RFC
Primary replicating enzyme Pol III Pol d/e
Clamp subunit of DNA polymeraseb
PCNA
Removes RNA Primer Pol I FEN-1Synthesizes RNA primer Primase Primase
Seals Okazaki fragments DNA ligase DNA ligase
Devlin, Textbook of Biochemistry, 2006
DNA SYNTHESIS: PROTEINS
GENE EXPRESSION 1
MAVillamo r, MD,MHPEd
Department of B iochemistry
Januar 2012
DNA SYNTHESIS
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DNA POLYMERASEFUNCTION E.Coli Eukaryote
Priming DnaG Pol a/ primase
Synthesis of continuous strand Pol III Pol d
Okazaki Fragment Initial Synthesis Pol a/ primase
Synthesis of Okazaki Fragment Pol III Pol d
Gap feeling after primer removal Pol I Pol dg
Damage bypass Pol IV,V Pol e x h i
Base excision repair Pol I Pol b
Mitochondrial DNA replication Pol g
Devlin, Textbook of Biochemistry, 2006
DNA SYNTHESIS
GENE EXPRESSION 1
MAVillamo r, MD,MHPEd
Department of B iochemistry
Januar 2012
TERMINATION: PRIMER REMOVAL
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TERMINATION: PRIMER REMOVAL
3
3
5
5
3
5
3
5
ATP
GENE EXPRESSION 1
MAVillamo r, MD,MHPEd
Department of B iochemistry
Januar 2012
DNA SYNTHESIS: TERMINATION Eukaryotes
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LINEAR GENOME
TELOMERES
Prevent recombination and shortening of
the lagging strand
Repeats of 6nucleotide, G-rich sequences
Humans : 5TTAGGG3
y
GENE EXPRESSION 1
MAVillamo r, MD,MHPEd
Department of B iochemistry
Januar 2012
DNA SYNTHESIS: TERMINATION Eukaryotes
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LINEAR GENOME
TELOMERASES
Add 6 nucleotide
repeats to 3OH end of
DNA
RNA serves as template
Reverse transcriptase
y
GENE EXPRESSION 1
MAVillamo r, MD,MHPEd
Department of B iochemistry
Januar 2012
DNA REPLICATION - SUMMARY
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Unwinding of dsDNA to provide
an ssDNA template
Formation of the replication fork
Initiation of DNA synthesis andelongation
Ligation of newly synthesizedDNA segments
Reconstitution of chromatinstructure.
GENE EXPRESSION 1
MAVillamo r, MD,MHPEd
Department of B iochemistry
Januar 2012
CHROMOSOME
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CHROMOSOME
chromatin
histones Nucleus
Cell Cycle
S phase
OriC
DNA
Replication
Semiconservative
Elongation
Stabilized by
To form
Located in
contains
undergoes
Described as
during
of the Initiatedby
Results
in
Template in
Ribosome
tRNA
rRNA
mRNA
Introns
ExonsPolymerization
Promoter
Translation
Transcription
Polypeptide
Starts in
producing
located
produces
producing
Composed
of
Templatein
by
Site of
REFERENCES
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Devlin, Thomas (2008). Textbook of
Biochemistry with Clinical Correlations. 7thEdition. Chapter 4, 139-176.
Murray,RK,Bender,D. ,Botham,K., Kennelly,
Rodwell,V. and Weil, A. (2009). HarpersIllustrated Biochemistry. International Ed.
Chapter 35 (312-334).
Nelson,D. and Cox,M. (2008). Lehninger
Principles of Biochemistry. Fifth Ed. Chapter 25.
Berg, et al. (2007). Biochemistry. Sixth Edition.
REFERENCES
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