watson and crick-1953

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Division Ave. High School Ms. Foglia AP Biology

AP Biology 2007-2008

DNA Replication

AP Biology

Watson and Crick-1953

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AP Biology

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

AP Biology

Directionality of DNA § You need to

number the carbons! u The 5’end of

the DNA strand will be near the phosphate group.

OH

CH2 O

4ʹ

5ʹ

3ʹ 2ʹ

1ʹ

PO4

N base

ribose

Nucleotide

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AP Biology

The DNA Backbone § Putting the DNA

backbone together u  refer to the 3ʹ and 5ʹ

ends of the DNA § New nucleotides are

added to the 3’ carbon of the sugar.

§  This will form a special covalent bond called a phosphodiester bond

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

AP Biology

Anti-Parallel Strands § Nucleotides in DNA

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

u complementary strand runs in opposite direction

3ʹ

5ʹ

5ʹ

3ʹ

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AP Biology

Bonding in DNA

3ʹ

5ʹ 3ʹ

5ʹ

covalent phosphodiester

bonds

hydrogen bonds

AP Biology

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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AP Biology

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 SEMI CONSERVATIVE

AP Biology

DNA Replication §  Large team of enzymes coordinates replication using

multiple origins.

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AP Biology

DNA Replication in Prokaryotes § One circular chromosome will form two

circular chromosomes.

AP Biology

Replication: 1st step § Unwind DNA using topoisomerase

u Helicase enzyme § Separates the DNA helix §  stabilized by single-stranded binding proteins

single-stranded binding proteins replication fork

helicase

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AP Biology

Replication: 2nd step § Build daughter DNA strand

u add new complementary bases

u DNA polymerase III u Energy comes from

hydrolyzing ATP

AP Biology

§ Adding bases u Can only add nucleotides to

3ʹ end of a growing DNA strand § One strand is called the LEADING STRAND, and

this process will happen continually as fast as the two strands separate.

u strand only grows 5ʹ→3ʹ

Replication

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AP Biology

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

ü

û

3ʹ

5ʹ

growing replication fork

AP Biology

DNA polymerase III

RNA primase u  Lays down an RNA primer 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

STEP 1-RNA Primase

5ʹ

5ʹ

5ʹ

3ʹ

3ʹ

3ʹ

5ʹ

3ʹ 5ʹ 3ʹ 5ʹ 3ʹ

growing replication fork primase

RNA

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AP Biology

DNA polymerase III

Occurs in both directions- The lagging strand will create pieces.

DNA polymerase III u  Copies DNA adding to the 3’

end

STEP 2-DNA Polymerase III Copies

5ʹ

5ʹ

5ʹ

3ʹ

3ʹ

3ʹ

5ʹ

3ʹ 5ʹ 3ʹ 5ʹ 3ʹ

growing replication fork primase

RNA

AP BiologyDNA Polymerase 1 swaps RNA primer for DNA

STEP 3-Replacing RNA primers with DNA

5ʹ

5ʹ

5ʹ

5ʹ

3ʹ

3ʹ

3ʹ

3ʹ


DNA polymerase I

RNA

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AP Biology

DNA Ligase will fuse the Okazaki Fragments to form a continuous strand.

STEP 4-Fuse the Okazaki Fragments

5ʹ

5ʹ

5ʹ

5ʹ

3ʹ

3ʹ

3ʹ

3ʹ


RNA

DNA ligase

AP Biology

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


DNA polymerase I

RNA

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AP Biology

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


TTAAGGG TTAAGGG TTAAGGG

AP Biology

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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AP Biology

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

AP Biology

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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AP Biology

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

watson and crick-1953

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