GE 1 DNA Synthesis 2011-12 HO

7/25/2019 GE 1 DNA Synthesis 2011-12 HO

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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


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


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


Januar 2012

CHROMOSOMAL COMPOSITION


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GENE EXPRESSION 1


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


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


Januar 2012


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CHEMISTRY: MITOCHONDRIAL DNA

GENE EXPRESSION 1


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


Januar 2012

CENTRAL DOGMA


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GENE EXPRESSION 1


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


Januar 2012

FUNDAMENTAL RULES - Replication


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alexandria.healthlibrary.ca

GENE EXPRESSION 1


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


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


Januar 2012


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GENE EXPRESSION 1


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


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


Januar 2012

INITIATION TOPOISOMERASE II


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INITIATION: TOPOISOMERASE II

GENE EXPRESSION 1


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


GENE EXPRESSION 1


Januar 2012

INITIATION HELICASE


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INITIATION: HELICASE

GENE EXPRESSION 1


Januar 2012

INITIATION HELICASE


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INITIATION: HELICASE

GENE EXPRESSION 1


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


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


Januar 2011

DNA SYNTHESIS: PRIMING

PRIMER SYNTHESIS


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GENE EXPRESSION 1


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


Januar 2011


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


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


Januar 2011

DNA SYNTHESIS:ELONGATION

DNA SYNTHESIS: ELONGATION


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GENE EXPRESSION 1


Januar 2011


5

3

3

5


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ELONGATION: DNA POLYMERASE


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ELONGATION: DNA POLYMERASE

GENE EXPRESSION 1


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


Januar 2012

DNA POLYMERASE III: SUBUNITS


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DNA POLYMERASE III: SUBUNITS

GENE EXPRESSION 1


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



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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


Januar 2012



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GENE EXPRESSION 1


Januar 2011




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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


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



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



Januar 2012

LIGATION


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LIGATION

GENE EXPRESSION 1



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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



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



Januar 2012

TERMINATION: PRIMER REMOVAL


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TERMINATION: PRIMER REMOVAL

3

3

5

5

3

5

3

5

ATP

GENE EXPRESSION 1



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



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



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



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

GE 1 DNA Synthesis 2011-12 HO

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