2007-2008 AP Biology

DNA Replication

Watson and Crick1953 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

Directionality of DNA You need to

number the carbons! it matters!

OH

CH2

O

4

5

3 2

1

PO4

N base

ribose

nucleotide

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

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

Bonding in DNA

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

3

5 3

5

covalentphosphodiester

bonds

hydrogenbonds

Base pairing in DNA Purines

adenine (A) guanine (G)

Pyrimidines thymine (T) cytosine (C)

Pairing A : T

2 bonds C : G

3 bonds

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

DNA Replication Large team of enzymes coordinates replication

DNA-pol of eukaryotes

DNA-pol : elongation DNA-pol III

DNA-pol : initiate replication and synthesize primers

DnaG, primase

DNA-pol : replication with low fidelity

DNA-pol : polymerization in mitochondria

DNA-pol : proofreading and filling gap

DNA-pol I

repairing

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

DNAPolymerase III

Replication: 2nd step Build daughter DNA

strand add new

complementary bases DNA polymerase III

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!

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

§2.2 Primase

• Also called DnaG

• Primase is able to synthesize primers using free NTPs as the substrate and the ssDNA as the template.

• Primers are short RNA fragments of a several decades of nucleotides long.

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

DNA polymerase III

Replication fork / Replication bubble

5

3 5

3

leading strand

lagging strand

leading strand

leading 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

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

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

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

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

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

1

2

3

4

What does it really look like?

Characteristics of replication

Semi-conservative replication

Bidirectional replication

Semi-continuous replication

High fidelity

§1.1 Semi-Conservative Replication

Semiconservative replication

Half of the parental DNA molecule is conserved in each new double helix, paired with a newly synthesized complementary strand. This is called semiconservative replication

Semiconservative replication

Experiment of DNA semiconservative replication

"Heavy" DNA(15N)

grow in 14N medium

The first generation

grow in 14N medium

The second generation

Significance

The genetic information is ensured to be transferred from one generation to the next generation with a high fidelity.

Bidirectional Replication

• Replication starts from unwinding the dsDNA at a particular point (called origin), followed by the synthesis on each strand.

• The parental dsDNA and two newly formed dsDNA form a Y-shape structure called replication fork.

3'

5'

5'

3'

5'

3'

5'3'

direction of replication

Replication fork

Bidirectional replication

• Once the dsDNA is opened at the origin, two replication forks are formed spontaneously.

• These two replication forks move in opposite directions as the syntheses continue.

Bidirectional replication

Replication of prokaryotes

The replication process starts from the origin, and proceeds in two opposite directions. It is named replication.

Replication of eukaryotes

• Chromosomes of eukaryotes have multiple origins.

• The space between two adjacent origins is called the replicon, a functional unit of replication.

origins of DNA replication (every ~150 kb)

Semi-continuous Replication

The daughter strands on two template strands are synthesized differently since the replication process obeys the principle that DNA is synthesized from the 5´ end to the 3´end.

5'

3'

3'

5'

5'

direction of unwinding3'

On the template having the 3´- end, the daughter strand is synthesized continuously in the 5’-3’ direction. This strand is referred to as the leading strand.

Leading strand

Semi-continuous replication

3'

5'

5'3'

replication direction

Okazaki fragment

3'

5'

leading strand

3'

5'

3'

5'replication fork

• Many DNA fragments are synthesized sequentially on the DNA template strand having the 5´- end. These DNA fragments are called Okazaki fragments. They are 1000 – 2000 nt long for prokaryotes and 100-150 nt long for eukaryotes.

• The daughter strand consisting of Okazaki fragments is called the lagging strand.

Okazaki fragments

Continuous synthesis of the leading strand and discontinuous synthesis of the lagging strand represent a unique feature of DNA replication. It is referred to as the semi-continuous replication.

Semi-continuous replication

2007-2008 AP Biology

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