AP Biology 2007-2008

DNA Replication

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Watson and Crick 1953 1953 article in Nature

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Directionality of DNA § You need to

number the carbons! u  it matters! u 3’ refers to

the 3 carbon on the sugar

u 5’ refers to the 5 carbon on the sugar.

OH

CH2 O

4ʹ

5ʹ

3ʹ 2ʹ

1ʹ

PO4

N base

ribose

nucleotide

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The DNA backbone § Putting the DNA

backbone together u  refer to sugar bonded

to the phosphate groups.

u The BASES are not the backbone.

OH

O

3ʹ

PO4

base

CH2 O

base

O P O

C

O –O

CH2

1ʹ

2ʹ

4ʹ

5ʹ

1ʹ

2ʹ

3ʹ

3ʹ

4ʹ

5ʹ

5ʹ

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Anti-parallel strands §  Nucleotides in DNA backbone

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

“direction” u One strand is 5’-3’ while the

other is 3’ to 5’ u Complementary strand

runs in opposite direction u Strands held together with

HYDROGEN BONDS. 3ʹ

5ʹ

5ʹ

3ʹ

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Bonding in DNA

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

3ʹ

5ʹ 3ʹ

5ʹ

covalent phosphodiester

bonds

hydrogen bonds

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Base pairing in DNA § Purines (2 rings)

u adenine (A) u guanine (G)

§ Pyrimidines (1 ring) u  thymine (T) u cytosine (C)

§ Pairing u A : T

§  2 bonds u C : G

§  3 bonds

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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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Copying DNA § Replication of DNA

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

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

u This is called SEMI-CONSERVATIVE

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DNA Replication §  Large team of enzymes coordinates replication §  Speeds up using multiple origins

Let’s meet the team…

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Slight differences in Prokaryotes

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Replication: 1st step § Unwind DNA with TOPOISOMERASE

u Helicase enzyme §  splits apart the DNA helix (break H-bonds) § Stabilized each piece using single-stranded

binding proteins

single-stranded binding proteins replication fork

helicase

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DNA Polymerase III

Replication: 2nd step

But… We’re missing something!

What?

Where’s the ENERGY

for the bonding!

§ Build daughter DNA strand u add new

complementary bases using.. DNA polymerase III

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energy

ATP GTP TTP CTP

Energy of Replication Where does energy for bonding usually come from?

ADP AMP GMP TMP CMP modified nucleotide

energy

We come with our own

energy!

And we leave behind a nucleotide!

You remember

ATP! Are there other ways

to get energy out of it?

Are there other energy nucleotides? You bet!

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Energy of Replication §  The nucleotides arrive as nucleosides

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

u  DNA bases arrive with their own energy source for bonding

u  bonded by enzyme: DNA polymerase III

ATP GTP TTP CTP

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§ Adding bases u can only add

nucleotides to 3ʹ end of a growing DNA strand §  need a “starter”

nucleotide to bond to

u strand only grows 5ʹ→3ʹ

DNA Polymerase III

DNA Polymerase III

DNA Polymerase III

DNA Polymerase III

energy

energy

energy

Replication energy

3ʹ

3ʹ

5ʹ B.Y.O. ENERGY! The energy rules

the process

5ʹ

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energy

3ʹ 5ʹ

5ʹ

5ʹ

3ʹ

need “primer” bases to add on to

energy

energy

energy

3ʹ

no energy to bond

energy

energy

energy

ligase

3ʹ 5ʹ

û

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

an existing DNA strand

Leading & Lagging strands

5ʹ

5ʹ

5ʹ

5ʹ

3ʹ

3ʹ

3ʹ

5ʹ 3ʹ

5ʹ 3ʹ 3ʹ

Leading strand

Lagging strand

Okazaki fragments

ligase

Okazaki

Leading strand u  continuous synthesis

Lagging strand u  Okazaki fragments u  joined by ligase

§ “spot welder” enzyme

DNA polymerase III

!

û

3ʹ

5ʹ

growing replication fork

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DNA polymerase III

Replication fork / Replication bubble

5ʹ

3ʹ 5ʹ

3ʹ

leading strand

lagging strand

leading strand

lagging strand leading strand

5ʹ

3ʹ

3ʹ

5ʹ

5ʹ

3ʹ 5ʹ

3ʹ

5ʹ

3ʹ 5ʹ

3ʹ

growing replication fork

growing replication fork

5ʹ

5ʹ

5ʹ

5ʹ

5ʹ 3ʹ

3ʹ

5ʹ

5ʹ lagging strand

5ʹ 3ʹ

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DNA polymerase III

RNA primer u  built by primase u  serves as starter sequence

for DNA polymerase III

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

an existing DNA strand

Starting DNA synthesis: RNA primers

5ʹ

5ʹ

5ʹ

3ʹ

3ʹ

3ʹ

5ʹ

3ʹ 5ʹ 3ʹ 5ʹ 3ʹ

growing replication fork primase

RNA

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DNA polymerase I u  removes sections of RNA

primer and replaces with DNA nucleotides

But DNA polymerase I still can only build onto 3ʹ end of an existing DNA strand

Replacing RNA primers with DNA

5ʹ

5ʹ

5ʹ

5ʹ

3ʹ

3ʹ

3ʹ

3ʹ

growing replication fork

DNA polymerase I

RNA

ligase

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

DNA polymerase III

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

Chromosome erosion

5ʹ

5ʹ

5ʹ

5ʹ

3ʹ

3ʹ

3ʹ

3ʹ

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 u  limit to ~50 cell divisions

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

§  high in stem cells & cancers -- Why?

telomerase

Telomeres

5ʹ

5ʹ

5ʹ

5ʹ

3ʹ

3ʹ

3ʹ

3ʹ

growing replication fork

TTAAGGG TTAAGGG TTAAGGG

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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

u 1000 bases/second! u main DNA builder

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

DNA polymerase III enzyme

Arthur Kornberg 1959

Roger Kornberg 2006

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Editing & proofreading DNA §  1000 bases/second =

lots of typos!

§  DNA polymerase I u  proofreads & corrects

typos u  repairs mismatched bases u  removes abnormal bases

§  repairs damage throughout life

u  reduces error rate from 1 in 10,000 to 1 in 100 million bases

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Fast & accurate! §  It takes E. coli <1 hour to copy

5 million base pairs in its single chromosome u divide to form 2 identical daughter cells

§ Human cell copies its 6 billion bases & divide into daughter cells in only few hours u  remarkably accurate u only ~1 error per 100 million bases u ~30 errors per cell cycle

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What does it really look like?

AP Biology 2007-2008

Any Questions??

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