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DNADNA

Filename DNA.pptFilename DNA.ppt Developed by D.ThomasDeveloped by D.Thomas Updated Oct 1999 by H.B. FackrellUpdated Oct 1999 by H.B. Fackrell

Material for Introductory Material for Introductory Microbiology 55-237Microbiology 55-237

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The quick

brown fox

jumps over

the lazy dog

Deoxy-ribo-

nucleic acid

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O O

Ribose

CH OH2

1'

2'3'

4'

5'

H

OH

OH O

H H H

H

Deoxy ribose

2'

H

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O BaseC

P

Pentose

Nucleoside O BaseC

Nucleotide

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CH2

NH2

N

OH OH

NN

N Adenine

RiboseO-P-O

O

O

Phosphate5'

The nucleotide,

adenosine 5’ monophosphate (AMP)

Nucleotide

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CH2

NH2

N

OH OH

O

NN

NAdenine

Ribose

Cousins of AMP.......

The energetic .... Adenosine triphosphate (ATP)

O-P-O

O

O

O-P-

O

O

O-P-

O

O

(Yet another nucleotide)

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TheThe event event ofTheThe event event of

20th Century biology20th Century biology

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The event of 20thC biologyThe event of 20thC biologyThe event of 20thC biologyThe event of 20thC biology

A paper published in Nature 171: 737-738A paper published in Nature 171: 737-738 Not a lengthy paper, but it contains the words Not a lengthy paper, but it contains the words

that started a revolution in biologythat started a revolution in biology Dateline Dateline 19531953 Authors, Authors, J.D. Watson and F.H.C. CrickJ.D. Watson and F.H.C. Crick Title: Title:

A structure for deoxyribose nucleic acidA structure for deoxyribose nucleic acid

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DNA strands are DNA strands are complementarycomplementary

POH P P P

P P POH

P

5’

5’

3’

3’

CC A T

ATGG

WWATSONATSON

KCIRKCIRCC

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Base*P - CH2

1'

2'3'

4'

O

OH H

2’ deoxynucleoside monophosphate

*A, T, G or C

Sugar

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POH P P P

P P POH

P

5’

5’

3’

3’

CC A T

ATGG

PHOSPHODIESTER BONDSPHOSPHODIESTER BONDS

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Base 1 Base 2(sugar)- 3’OH HO-P-O-(sugar)+

Base 1

(sugar)- -(sugar)

O

O

O-P-O + H2O

O

Phosphodiester bond

O

Base 2

-3’OH

-3’OH

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POH P P P

P P POH

P

5’

5’

3’

3’

CC A T

ATGG

Complementary strands interact by Complementary strands interact by hydrogen bonding of specific base pairshydrogen bonding of specific base pairs

A=CA=C G=CG=C

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NN

N

H

HH

CH

1’

CH3O

H

O

NH

N

O

NC

N HH

H

Base pairing

Adenine Thymine

Guanine Cytosine

1’

1’

1’

1’ 1’ refers to the 1’ carbon of deoxyribose

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Purine

PyrimidineNN

N

H

HH

CH

1’

CH3O

Hydrogen bonding

A T=

1’

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H

O

NH

N

O

NC

N HH

H

Hydrogen bonding

GG CC

1’1’

1’1’

Purine Pyrimidine

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In summary...

A T

CG

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Each strand has a free Each strand has a free 5’5’ end and a free end and a free 3’3’ end end

Strands are Strands are antiparallelantiparallel

POH P P P

P P POH

P

5’

5’

3’

3’

CC A T

ATGG

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Strands hydrogen bonded by specific base Strands hydrogen bonded by specific base pairing (A loves T; G C!)pairing (A loves T; G C!)

Strands are complementaryStrands are complementary

POH P P P

P P POH

P

5’

5’

3’

3’

CC A T

ATGG

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Base

Deoxyribose (sugar)

1'2'3'

4'

5'

P =

O

O

O

OP

c5

c5

c5

P

P

P

OH

BaseSugar

Phosphate

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Base

1'2'3'

4'

5'

c5

c5

c5

P

P

P

OH Bases form the “rungs” of the DNA ladder

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1'2'3'

4'

5'

c5

c5

c5

P

P

P

OH Sugar-phosphates form each “upright” of the DNA ladder

SugarSugar

SS

SS

SS

S

SPS

P

P

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1'2'3'

4'

5'

c5

c5

C5

P

P

P

OH

5’ end

3’end

The The 5’ end5’ end and and

the the 3’end 3’end are are differentdifferent

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DNA replication requiresDNA replication requires

DNA DNA templatetemplate PrimerPrimer on which to add new nucleotides on which to add new nucleotides DNA polymerase (enzyme)DNA polymerase (enzyme) NucleotidesNucleotides

– deoxy adenosine triphosphate (dATP)deoxy adenosine triphosphate (dATP)

– dGTPdGTP

– dCTPdCTP

– dTTPdTTP

ATP, MgATP, Mg++++

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DNA replicationDNA replication

TemplateTemplate determines determines whichwhich nucleotide to add next nucleotide to add next

3’OH3’OH

Primer Primer determines determines wherewhere the next nucleotide is the next nucleotide is addedadded

The next nucleotideThe next nucleotide

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Base*P - CH2

1'

2'3'

4'

O

OH H

3’-OH where the next nucleotide will be added

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GrowthGrowth of a nucleic acid of a nucleic acid

P OHPPPPP5’

3’B1 B3B2 B4

OH3’

B5

PPP5’

PPPPPP5’

3’B1 B3B2 B4

OH3’

B5

+ PP5’

P

Growth is from 5’ to 3’ end

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POH P P P

P

P OH

P

5’

5’

3’

CC

A

CTGG

3’ OH

Template Template determines determines whatwhat the next base will be (A) the next base will be (A)

Primer 3’OH is Primer 3’OH is wherewhere the next base will go the next base will go

Next baseNext base

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Replication “eye” or “bubble”

Double-stranded (ds DNA)

Origin of replication

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DNA replicationDNA replication

Replication Replication “fork“fork” in one corner of “eye”” in one corner of “eye”

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DNA replicationDNA replication

Parent strandsParent strands Daughter strandsDaughter strands

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DNA replicationDNA replication

5’

5’

3’

3’ 5’

3’

5’

3’

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<- As the eye enlarges...<- As the eye enlarges...

5’

5’

3’

3’5’

3’

5’

3’

5’

This strand, the leading strand, can

elongate freely. DNA synthesis is

continuous towards corner of “eye”

This strand, the lagging strand, grows as fragments which are later ligated together

3’

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Okazaki fragments (DNA)Okazaki fragments (DNA)

5’

5’

3’

3’5’

3’

5’

3’

5’

Leading strand (continuous strand)

Lagging or discontinuous strand Fragments, before they ligate together are named after their discoverer, Okazaki

3’

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Replication involves manyReplication involves many origins origins

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Replication of DNA needs Replication of DNA needs several enzymesseveral enzymes

HelicaseHelicase (to unwind the alpha helix) (to unwind the alpha helix) PrimasePrimase (to build RNA primer segments) (to build RNA primer segments) DNA polymeraseDNA polymerase (to extend primers, and (to extend primers, and

more)more) DNA ligaseDNA ligase (to link together the ends of (to link together the ends of

adjacent DNA fragmentsadjacent DNA fragments

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Replication of DNA in briefReplication of DNA in brief (5 steps)(5 steps)

ss DNA

RNA primerRNA primer

1) synthesis of 1) synthesis of RNARNA primer (evolutionary implications?) primer (evolutionary implications?)

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Replication of DNA in briefReplication of DNA in brief (5 steps)(5 steps)

ss DNA (old)

RNA primerRNA primer

2) 2) New DNA added to added to RNARNA primer by DNA primer by DNA

polymerase polymerase

3’3’ 5’5’

3’3’5’5’

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Replication of DNA in briefReplication of DNA in brief (5 steps)(5 steps)

ss DNA (old)

3) 3) RNARNA primer cleaved out (by DNA polymerase) primer cleaved out (by DNA polymerase)

3’3’ 5’5’

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Replication of DNA in briefReplication of DNA in brief (5 steps)(5 steps)

ss DNA (old)

4) 4) New DNA synthesized (DNA polymerase) where RNA synthesized (DNA polymerase) where RNA primer cleaved outprimer cleaved out

3’3’ 5’5’

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Replication of DNA in briefReplication of DNA in brief (5 steps)(5 steps)

ss DNA (old)

5) 5) DNA ligase joins 3’ end of each newly completed fragment with 5’ end of the next to give:

3’3’ 5’5’

3’ 5’3’ 5’

parent strand

new complementary strand

3’3’5’5’

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PP OHOHAA A’A’

Given, DNA, single stranded in the middle, ds at endsGiven, DNA, single stranded in the middle, ds at ends

1. For the B segment, which end, B or B’ represents the 3’ 1. For the B segment, which end, B or B’ represents the 3’ end?end?

BB B’B’

2.. For the A fragment, which end, A or A’ represents the 3’ 2.. For the A fragment, which end, A or A’ represents the 3’ end?end?

3.. If we fill the gap in a test tube (in vitro) reaction containing 3.. If we fill the gap in a test tube (in vitro) reaction containing only NTP’s and DNA polymerase, how many pieces will the only NTP’s and DNA polymerase, how many pieces will the lower strandlower strand contain? contain?

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In the lab, DNA strands can be separated and re-annealed

Heat, alkaliHeat, alkali

Cool, neutral pHCool, neutral pH

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DNA strands can be separated and re-annealed

Amazing/ It must be Amazing/ It must be

our destiny!our destiny!

““By Jove, if it isn’t....”By Jove, if it isn’t....”

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DNA strands can be separated and re-annealed

• • IImportantmportant in in nature nature

• • IImportant mportant in in biological sciencesbiological sciences

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The quiCk

brown fox jumps over

The lAzy doG