DNA Replication: Mechanisms, Enzymes, and Processes, Slides of Genetics

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DNA REPLICATION

^

Genetic information is transferred fromparents to progeny organisms/cells by afaithful replication of the parental DNAmolecules. ^

The prime role of any mode of replicationis to duplicate the base sequence of theparent molecule.

REPLICONS

•^

Replicon is any piece of DNA whichreplicates as a single unit.

-^

It contains an origin and sometimes aterminus.

-^

Origin is the DNA sequence where areplicon initiates its replication.

-^

Terminus is the DNA sequence where areplicon usually stops its replication

REPLICATION OF A CIRCULAR

BACTERIAL REPLICON

-^

All prokaryotic chromosomes and manybacteriophage and viral DNA molecules arecirclular and comprise single replicons.

-^

There is a single termination site roughly 180

o

opposite the unique origin.

-^

The long, linear DNA molecules of eukaryoticchromosomes consist of mutiple regions, eachwith its own orgin.

-^

A typical mammalian cell has 50000-100000replicons with a size range of 40-200 kb.

MULTIPLE EUKARYOTIC REPLICONS

AND REPLICATION BUBBLES

1. dNTPs: dATP, dTTP, dGTP, dCTP

(deoxyribonucleoside 5’-triphosphates)(sugar-base + 3 phosphates)

2. DNA template3. DNA polymerase I (formerly the

Kornberg

enzyme

(DNA polymerase II & III discovered soonafter)

4. Mg

2+

(optimizes DNA polymerase activity)

FOUR COMPONENTS ARE REQUIRED

THE ENZYMOLOGY

OF DNA

REPLICATION

^

In 1957, Arthur Kornberg demonstrated theexistence of a DNA polymerase - DNApolymerase I ^

DNA Polymerase I has THREE differentenzymatic activities in a single polypeptide: ^

a 5’ to 3’ DNA polymerizing activity ^

a 3’ to 5’ exonuclease activity ^

a 5’ to 3’ exonuclease activity

Subsequenthydrolysis ofPPi drives thereaction forward^ Nucleotides are added at the 3'-end of the strand

THE 5’ TO 3’ DNA POLYMERIZING ACTIVITY

Proof reading activityof the 3’ to 5’ exonuclease. •^

DNAPI stalls if the incorrectnucleotide is added

-^

it can’t add the next nucleotide inthe chain Proof reading activity isslow as compared to polymerizingactivity, but the stalling of DNAP Iafter insertion of an incorrect baseallows the proofreading activity tocatch up with the polymerizingactivity and remove the incorrectbase.

How?1) Base-pairing specificity at the active site- correct geometry in the active site occurs only withcorrectly paired bases BUT the wrong base still getsinserted 1/ 10

4 -

5 dNTPs added

2. Proofreading activity by 3’-5’ exonuclease- removes mispaired dNTPs from 3’ end of DNA- increases the accuracy of replication 10

2 -

3 fold

3. Mismatch repair system- corrects mismatches AFTER DNA replication

DNA Replication is Accurate(In E. coli: 1 error/

9 -

10 dNTPs added)

Is DNA Polymerase I the principal

replication enzyme??

^

In 1969 John Cairns and Paula deLucia

isolated a

mutant bacterial strain with only 1% DNAP Iactivity (polA) ^

mutant was super sensitive to UV radiation ^

but otherwise the mutant was fine i.e. it coulddivide, so obviously it can replicate its DNA ^

Conclusion: ^

DNAP I is NOT the principal replication enzymein E. coli

• DNAP I is too slow (600 dNTPs added/minute – would

take 100 hrs to replicate genome instead of 40minutes)

• DNAP I is only moderately processive

(processivity refers to the number of dNTPs added toa growing DNA chain before the enzyme dissociatesfrom the template) Conclusion: ^

There must be additional DNA polymerases. ^

Other clues…. Biochemists purified them from the polA mutant

A total of 5 different DNAPs have been

reported in E. coli

^

DNAP I: functions in repair and replication

^

DNAP II: functions in DNA repair

(proven in 1999)

^

DNAP III: principal DNA replication enzyme

^

DNAP IV: functions in DNA repair

(discovered in

^

DNAP V: functions in DNA repair

(discovered in

The DNA Polymerase Family 1999)

The "real" replicative polymerase in E. coli

^

It’s fast: up to 1,000 dNTPs added/sec/enzyme ^

It’s highly processive: >500,000 dNTPs added beforedissociating ^

It’s accurate: makes 1 error in 10

7 dNTPs added, with

proofreading, this gives a final error rate of 1 in 10

10

DNA Polymerase III overall.