AP Biology 2007-2008

Synthesis of DNA

June.21.2010

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DNA synthesis occurs by the process of replication.

During replication, each of the two parental strands of DNA serves as a template for the synthesis of aComplementary strand.

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Each molecule generated by the replication process contains

one intact parental strand and one newly synthesized strand.

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In eukaryotes, DNA replication occurs during the S phase of the cell cycle

The cell divides during the next phase (M), and each daughter cell receives an exact copy of the DNA of the parent cells.

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Cultured MDCK cells

Day 1 Day 3

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Watson and Crick1953 article in Nature

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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

Sounds trivial, but…this will be

IMPORTANT!!

OH

O

3

PO4

base

CH2

O

base

OPO

C

O–O

CH2

1

2

4

5

1

2

3

3

4

5

5

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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 direction

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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Base pairing in DNA Purines

adenine (A) guanine (G)

Pyrimidines thymine (T) cytosine (C)

Pairing A : T

2 bonds C : G

3 bonds

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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

single-stranded binding proteins replication fork

helicase

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Definitions Template strands: The old strands

which are now separated from each other.

New strands: The growing complementary nucleotide sequences that allow for forming the two double helix structures

Note that replication is direction specific.

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DNA Elongation Works only 5’ to 3’ This means that the 5’ end of

the nucleotide links on to the 3’ end of the DNA (or RNA primer)

The NEW strand only grows 5’ to 3’

OH

O

3

PO4

base

CH2

O

base

OPO

C

O–O

CH2

1

2

4

5

1

2

3

3

4

5

5

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DNA polymerase III

RNA primer built by primase serves as starter sequence

for DNA polymerase III

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

an existing DNA strand

Starting DNA synthesis: RNA primers

5

5

5

3

3

3

5

3 53 5 3

growing replication fork

primase

RNA

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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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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 polymerase III

Replication fork / Replication bubble

5

3 5

3

leading strand

lagging strand

leading strand

lagging strandleading strand

5

3

3

5

5

3

5

3

5

3 5

3

growing replication fork

growing replication fork

5

5

5

5

53

3

5

5lagging strand

5 3

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DNAPolymerase III

Replication: 2nd step

But…We’re missing

something!What?

Where’s theENERGY

for the bonding!

Build daughter DNA strand add new

complementary bases DNA polymerase III

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DNA polymerase I removes sections of RNA

primer and replaces with DNA nucleotides

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

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

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

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

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1

2

3

4

What does it really look like?

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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

bonded by enzyme: DNA polymerase III

ATP GTP TTP CTP

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Adding bases can only add

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

nucleotide to bond to

strand only grows 53

DNAPolymerase III

DNAPolymerase III

DNAPolymerase III

DNAPolymerase III

energy

energy

energy

Replication energy

3

3

5B.Y.O. ENERGY!

The energy rulesthe process

5

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energy

35

5

5

3

need “primer” bases to add on to

energy

energy

energy

3

no energy to bond

energy

energy

energy

ligase

3 5

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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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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

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Editing & proofreading DNA 1000 bases/second =

lots of typos!

DNA polymerase I proofreads & corrects

typos repairs mismatched bases removes abnormal bases

repairs damage throughout life

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

AP Biology 2007-2008

Any Questions??

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energy

ATPGTPTTPATP

Energy of ReplicationWhere does energy for bonding usually come from?

ADPAMPGMPTMPAMPmodified nucleotide

We comewith our own

energy!

And weleave behind a

nucleotide!

Youremember

ATP!Are there other ways

to get energyout of it?

AP Biology

Adding bases can only add

nucleotides to 3 end of the growing DNA strand need a primer

nucleotide to bond to

strand grows 53

DNAPolymerase III

Replication energy

3

3

5B.Y.O. ENERGY!

The energy rulesthe process

5

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5

3

3

5

35

3 5

no energyto bond

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energy

5

3

3

5

35

3 5

ligase

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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

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

Houston, we have a problem!

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Replication fork

3’

5’

3’

5’

5’

3’

3’ 5’

direction of replication

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DNA synthesis in prokaryotes

1. Replication is bidirectional.

2. Replication is semi-conservative.

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Bidirectional replication of a circular chromosome

Replication begins at the point of origin (oriC) and proceeds in both directions at the same time.

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Unwinding of Parental Strands

Topoisomerases: can break phosphodiester bonds and rejoin them relieve the supercoiling of the parental duplex caused by unwinding.

DNA gyrase is a major topoisomerase in bacterial cells.

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以上です。