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DNA Replication

The Specifics

- Before DNA replication can occur, highly coiled and condensed DNA molecule must be “straightened” into linear sequence of nucleotidesEnzymes serve this function.- Enzymes serve this function

DNA ReplicationThe Specifics

DNA Replication

DNA helicase

replication fork

Step 1: As DNA uncoils and unwinds, a class of enzymes called topoisomerases relieve tension on molecule

Step 2: DNA helicase breaks hydrogen bonds that hold two strands together - produces a “replication fork”

Topoisomerase

DNA Replication

Step 4: RNA primase attaches RNA nucleotides (primers) to 3´ end of each strand

RNA primers

3´

5´

3´5´

Single stranded binding proteins (SSBP) attach to each strand to prevent hydrogen bonds from reforming

Step 3:

SSBP

DNA ReplicationStep 5:

3´

5´

3´5´

DNA polymerase III5´

3´

5´

3´

Starting at primers DNA polymerase III adds complimentary DNA nucleotides to each strand

DNA polymerase III constructs new strand in 5

´-3´ direction only

DNA Replication3´

5´

3´5´

NOTICE:One strand (leading) is built continuously towards replication fork

Continuou

s

Other strand (lagging) is built discontinuously away from replication fork

Discontinuous

5´

3´5´

3´

Okazaki fragmentsLagging strand is made up

of short pieces called Okazaki fragments

DNA polymerase I replaces RNA primers with DNADNA ligase joins the Okazaki fragments.

Okazaki fragments

DNA Replication3´

5´

3´5´

Step 6:

DNA ligase

DNA ligase joins Okazaki fragments DNA polymerase I

Error Correction:Most occurs during Step 5 when DNA polymerase III is adding complimentary DNA nucleotides

Step 7:

T C

DNA Replication

T

AT CGAG GATG GCCCT

GACTC

3´5´5´3´ ACCGGGA

New strand is made from 5’ to 3’

1000 nucleotides added per second in bacteria

50 nucleotides added per second in Eukaryotes

When this happens, enzyme usually backs up and corrects error, before continuing on

Step 7: Error Correction:Most occurs during Step 5 when DNA polymerase III is adding complimentary DNA nucleotidesOccasionally, DNA polymerase III will make a mismatch error

DNA Replication

C T

AT CGAG GTG GCCCT

GACTC

3´5´5´3´ ACCGGGA

A

G

Uncorrected Mismatch Error

Step 7: Error Correction:Most occurs during Step 5 when DNA polymerase III is adding complimentary DNA nucleotidesOccasionally, DNA polymerase III will make a mismatch error

About one error in a million base pairs is not corrected by DNA polymerase III

When this happens, enzyme usually backs up and corrects error, before continuing on

DNA Replication

C CAC

T

AT CGAG GTG GCCCT

GACT

3´5´5´3´ CGGGA

G

A

Special DNA repair complexes made up of proteins and enzymes, including DNA polymerases I and DNA polymerase II, remove a small section of nucleotides around error

Step 7:

C

DNA Replication

CAC T

AT CGAG GTG GCCCT

GACT

3´5´5´3´ CGGGA

A

T

Special DNA repair complexes made up of proteins and enzymes, including DNA polymerases I and DNA polymerase II, remove a small section of nucleotides around error

Step 7:

Missing section is replaced by DNA polymerase IIId by DNA ligase.

C

DNA Replication

CAC T

AT CGAG GTG GCCCT

GACT

3´5´5´3´ CGGGA

A

T

This phosphodiester bond is missing

Missing phosphodiester bond of last nucleotide is joined by DNA ligase

Special DNA repair complexes made up of proteins and enzymes, including DNA polymerases I and DNA polymerase II, remove a small section of nucleotides around error

Step 7:

Missing section is replaced by DNA polymerase IIId by DNA ligase.