2007-2008 AP Biology

DNA Replication

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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

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Directionality of DNA You need to

number the carbons! it matters!

OH

CH2

O

4

5

3 2

1

PO4

N base

ribose

nucleotide

This will beIMPORTANT!!

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The DNA backbone Putting the DNA

backbone together refer to the 3 and 5

ends of the DNA the last trailing carbon

OH

O

3

PO4

base

CH2

O

base

OPO

C

O–O

CH2

1

2

4

5

1

2

3

3

4

5

5

Sounds trivial, but…this will be

IMPORTANT!!

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Anti-parallel strands Nucleotides in DNA

backbone are bonded from phosphate to sugar between 3 & 5 carbons DNA molecule has

“direction” complementary strand runs

in opposite directionTHIS WILL CAUSE A

PROBLEM FOR REPLICATION

3

5

5

3

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Bonding in DNA

….strong or weak bonds?How do the bonds fit the mechanism for copying DNA?

3

5 3

5

covalentphosphodiester

bonds

hydrogenbonds

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Copying DNA Replication of DNA

base pairing allows each strand to serve as a template for a new strand

new strand is 1/2 parent template & 1/2 new DNA

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DNA Replication Large team of enzymes coordinates replication

Let’s meetthe team…

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Replication: 1st step Unwind DNA

helicase enzyme unwinds part of DNA helix stabilized by single-stranded binding proteins

PREVENTS DNA MOLECULE FROM CLOSING!

DNA gyrase Enzyme that prevents tangling upstream from the replication

fork

single-stranded binding proteins replication fork

helicase gyrase

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Replication: 2nd step Add RNA primer

DNA BY RNA Primase Why must this be done?

DNA can’t be added to an existing strand of nucleotides

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DNAPolymerase III

Replication: 3rd step

But…We’re missing

something!What?

Where’s theENERGY

for the bonding!

Build daughter DNA strand add new complementary

bases With the help of the

enzyme DNA polymerase III

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energy

ATPGTPTTPCTP

Energy of ReplicationWhere does energy for bonding usually come from?

ADPAMPGMPTMPCMPmodified nucleotide

energy

We comewith our own

energy!

And weleave behind a

nucleotide!

Youremember

ATP!Are there other ways

to get energyout of it?

Are thereother energynucleotides?

You bet!

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Energy of Replication The nucleotides arrive as nucleosides

DNA bases with P–P–P P-P-P = energy for bonding

DNA bases arrive with their own energy source for bonding: by breaking off two phosphate groups

bonded by enzyme: DNA polymerase III

ATP GTP TTP CTP

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4th step Replacement of RNA primer by DNA Done by DNA polymerase I

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DO NOW Write the steps of DNA replication

include all enzymes used and what they do!

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Before we solve the Problem. Lets review DNA replication

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DNA polyermase III can only add nucleotides to an existing strand

DNA polymerase III can only add nucleotides to 3 end of a DNA strand WHY IS THAT A PROBLEM?

Limits of DNA polymerase III

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Limits of DNA polymerase III can only build onto 3 end of

an existing DNA strand

Leading & Lagging strands

5

5

5

5

3

3

3

53

53 3

Leading strand

Lagging strand

Okazaki fragments

ligase

Okazaki

Leading strand continuous synthesis

Lagging strand Okazaki fragments joined by ligase

“spot welder” enzyme

DNA polymerase III

3

5

growing replication fork

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DNA polymerase III

RNA primer is added built by primase serves as starter sequence for DNA polymerase IIIHOWEVER short segments called Okazaki fragments

are made because it can only go in a 5 3 direction

DNA replication on the lagging strand

5

5

5

3

3

3

5

3 53 5 3

growing replication fork

primase

RNA

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NEXT DNA polymerase I removes sections of RNA

primer and replaces with DNA nucleotides

STRANDS ARE GLUED TOGETHER BY DNA LIGASE

Replacing RNA primers with DNA

5

5

5

5

3

3

3

3

growing replication fork

DNA polymerase I

RNA

ligase

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Lagging strand DNA replication review

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Loss of bases at 5 ends in every replication chromosomes get shorter with each replication limit to number of cell divisions?

DNA polymerase III

All DNA polymerases can only add to 3 end of an existing DNA strand

Chromosome erosion

5

5

5

5

3

3

3

3

growing replication fork

DNA polymerase I

RNA

Houston, we have a problem!

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Repeating, non-coding sequences at the end of chromosomes = protective cap limit to ~50 cell divisions

Telomerase enzyme extends telomeres can add DNA bases at 5 end different level of activity in different cells

high in stem cells & cancers -- Why?

telomerase

Telomeres

5

5

5

5

3

3

3

3

growing replication fork

TTAAGGGTTAAGGGTTAAGGG

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Replication fork

3’

5’

3’

5’

5’

3’

3’ 5’

helicase

direction of replication

SSB = single-stranded binding proteins

primase

DNA polymerase III

DNA polymerase III

DNA polymerase I

ligase

Okazaki fragments

leading strand

lagging strand

SSB

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DNA polymerases DNA polymerase III

1000 bases/second! main DNA builder

DNA polymerase I 20 bases/second editing, repair & primer removal

DNA polymerase III enzyme

Arthur Kornberg1959

Roger Kornberg2006