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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