DNA Replication Ch. 16 Watson and Crick 1953 article in Nature.

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DNA Replication Ch. 16

Transcript of DNA Replication Ch. 16 Watson and Crick 1953 article in Nature.

Page 1: DNA Replication Ch. 16 Watson and Crick 1953 article in Nature.

DNA ReplicationCh. 16

Page 2: DNA Replication Ch. 16 Watson and Crick 1953 article in Nature.

Watson and Crick1953 article in Nature

Page 3: DNA Replication Ch. 16 Watson and Crick 1953 article in Nature.

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

Page 4: DNA Replication Ch. 16 Watson and Crick 1953 article in Nature.

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!!

Page 5: DNA Replication Ch. 16 Watson and Crick 1953 article in Nature.

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!!

Page 6: DNA Replication Ch. 16 Watson and Crick 1953 article in Nature.

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

Page 7: DNA Replication Ch. 16 Watson and Crick 1953 article in Nature.

Bonding in DNA

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

3

5

3

5

covalentphosphodieste

rbonds

hydrogen

bonds

Page 8: DNA Replication Ch. 16 Watson and Crick 1953 article in Nature.

Base pairing in DNA

• Purines– adenine (A)– guanine (G)

• Pyrimidines– thymine (T)– cytosine (C)

• Pairing– A : T • 2 bonds

– C : G• 3 bonds

Page 9: DNA Replication Ch. 16 Watson and Crick 1953 article in Nature.

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• semi-conservative

copy process

Page 10: DNA Replication Ch. 16 Watson and Crick 1953 article in Nature.

DNA Replication

• Large team of enzymes coordinates replication

Let’s meetthe team…

Page 11: DNA Replication Ch. 16 Watson and Crick 1953 article in Nature.

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

I’d love to behelicase & unzip

your genes…

Page 12: DNA Replication Ch. 16 Watson and Crick 1953 article in Nature.

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

Page 13: DNA Replication Ch. 16 Watson and Crick 1953 article in Nature.

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!

Page 14: DNA Replication Ch. 16 Watson and Crick 1953 article in Nature.

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

Page 15: DNA Replication Ch. 16 Watson and Crick 1953 article in Nature.

• 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

Replicationenergy

3

3

5B.Y.O. ENERGY!

The energy rulesthe process

5

Page 16: DNA Replication Ch. 16 Watson and Crick 1953 article in Nature.

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

Page 17: DNA Replication Ch. 16 Watson and Crick 1953 article in Nature.

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

Page 18: DNA Replication Ch. 16 Watson and Crick 1953 article in Nature.

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

Page 19: DNA Replication Ch. 16 Watson and Crick 1953 article in Nature.

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

Page 20: DNA Replication Ch. 16 Watson and Crick 1953 article in Nature.

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!

Page 21: DNA Replication Ch. 16 Watson and Crick 1953 article in Nature.

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

Page 22: DNA Replication Ch. 16 Watson and Crick 1953 article in Nature.

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

Page 23: DNA Replication Ch. 16 Watson and Crick 1953 article in Nature.

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

Thomas Kornberg??

Page 24: DNA Replication Ch. 16 Watson and Crick 1953 article in Nature.

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

Page 25: DNA Replication Ch. 16 Watson and Crick 1953 article in Nature.

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 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

Page 26: DNA Replication Ch. 16 Watson and Crick 1953 article in Nature.

1

2

3

4

What does it really look like?

Page 27: DNA Replication Ch. 16 Watson and Crick 1953 article in Nature.

What Does It Really Look Like, (3D Animated Movie Version)

Page 28: DNA Replication Ch. 16 Watson and Crick 1953 article in Nature.
Page 29: DNA Replication Ch. 16 Watson and Crick 1953 article in Nature.

2007-2008

Any Questions??

Page 30: DNA Replication Ch. 16 Watson and Crick 1953 article in Nature.

Review Questions

Page 31: DNA Replication Ch. 16 Watson and Crick 1953 article in Nature.

Base your answers to the following questions on the choices below:

A. HelicaseB. PolymeraseC. LigaseD. Primase

1. Brings together the Okazaki fragments.2. Adds nucleotides to the leading strand.3. Recognizes the origin of replication in the DNA.

Page 32: DNA Replication Ch. 16 Watson and Crick 1953 article in Nature.

4. In DNA synthesis, DNA isA. Read 3’ to 5’ and made 5’ to 3’B. Read 3’ to 5’ and made 3’ to 5’C. Read 5’ to 3’ and made 3’ to 5’D. Read 5’ to 3’ and made 5’ to 3;