Maintenance and expression of genetic information

Central Dogma:

DNA RNA Protein

GAATTGCGCCTTTTG

5’-GAATTGCGCCTTTTG-33’-CTTAACGCGGAAAAC-5’

Minor Groove

Major Groove

DNA can be supercoiled

Semi-conservativeReplication of DNA

The Watson-Crick Model

Proof of the Watson-Crick Model:The Meselson-Stahl Experiment

The Meselson-Stahl Experiment # generations

0

0.3

0.7

1

1.1

1.5

1.9

2.3

3

4.1

0 and 1.9 mixed

0 and 4.1 mixed

Starting DNA Heavy/Heavy

1st generation All Heavy/Light

2nd generation Two Heavy/Light Two Light/Light

3rd generation Two Heavy/Light Six Light/Light

The Meselson-Stahl Experiment

DNA Polymerase

A 3’ hydroxylgroup is necessaryfor addition of nucleotides

1’

2’

3’

1’2’

3’4’

5’

4’5’

2’

3’

2’

3’4’

5’

4’5’

1’

DNA chain growth is driven by PPi release/hydrolysis

Accuracy of DNA polymerases is essential.

--Error rate is less than 1 in 108

--Due in part to “reading” of complementary bases

--also contains its own proofreading activity

DNA Polymerase contains a Proofreading subunit

Proofreading by DNA polymerase

Proofreading by DNA polymerase

Both Template strands are copied at a Replication Fork

The polarity of DNA synthesis creates an asymmetry between the leading strand and the lagging strand at the replication fork

Topoisomerase

Protein complexes of the replication fork

Protein complexes of the replication fork:DNA polymeraseDNA primaseDNA HelicasessDNA binding proteinSliding ClampClamp LoaderDNA LigaseDNA Topoisomerase

DNA primasesynthesizes anRNA primerto initiate DNAsynthesis on thelagging strand

Replication of the Lagging Strand

DNA ligase seals nicks left by lagging strand replication

DNA helicase unwinds the DNA duplexahead of DNA polymerasecreating single strandedDNA that can be usedas a template

DNA helicase moves along one strand of the DNA

ssDNA binding proteins are required to “iron out” the unwound DNA

ssDNA binding proteins bind to the sugar phosphate backboneleaving the bases exposed for DNA polymerase

DNA polymerase is not very processive(ie it falls off the DNAeasily). A “sliding clamp”is required to keep DNA polymerase on andallow duplication of longstretches of DNA

A “clamp loader:” complex is required to get the clamp ontothe DNA

Lagging strand synthesis

MCM proteins

PCNA

RPC

Topoisomerase

Ahead of the replication fork the DNA becomessupercoiled

The supercoiling aheadof the fork needs to be relieved or tension wouldbuild up (like coiling asspring) and block forkprogression.

Supercoiling is relieved by the action of Topoisomerases.

Type I topoisomerases:Make nicks in one DNA strandsCan relieve supercoiling

Type II topoisomersasesMake nicks in both DNA strands (double strand break)Can relieve supercoiling and untangle linked DNA helices

Both types of enzyme form covalent intermediates with the DNA

Topoisomerase IAction

Topoisomerase IAction

Topoisomerase IIAction

Topoisomerase IIAction

Topoisomerases as drug targets:

Because dividing cells require greater topoisomerase activitydue to increased DNA synthesis, topoisomerase inhibitors areused as chemotherapeutic agents.

E.g. Camptothecin -- Topo I inhibitor Doxorubicin -- Topo II inhibitor

These drugs act by stablilzing the DNA-Topoisomerase complex.

Also, some antibiotics are inhibitors of the bacterial-specifictoposisomerase DNA gyrase e.g. ciprofloxacin

DNA is replicated during S phase of the Cell Cycle

In S phase, DNA replication begins at origins of replication that are spread out across the chromosome

Each origin of replicatonInitiates the formationof bidirectionalreplication forks

Origins or replication are strictly controlled so that they “fire” only once per cell cycle

Errors lead to overreplication of specific chromosomal regions.(= gene amplification)

This seen commonly in cancer cells and can be an importantprognostic indicator.

It can also contribute to acquired drug resistance.E.g. Methotrexate induces amplification of theDihydrofolate Reductase locus.

Errors of DNA Replication and Disease

The rate of misincorporation of bases by DNA polymerase isextremely low, however repeated sequences can cause problems.

In particular, trinucleotide repeats cause difficulties which can lead to expansion of these sequences.

Depending where the repeat is located expansion of the sequencecan have severe effects on the expression of a gene or thefunction of a protein.

Several mechanisms for the expansion of trinucleotide repeatshave been proposed, but the precise mechanism is unknown.

From Stryer: Looping out of repeats before replication.

Several inherited diseases are associated with expansion of trinucleotide repeat sequences.

Very different disorders, but they share the characteristic of becoming more severe in succeeding generations due to progressiveexpansion of the repeats