DNA
E. McIntyre
IB Biology HL
DNA is the Genetic Material
• Therefore it must
• Replicate faithfully.
• Have the coding capacity to generate proteins and other products for all cellular functions.
“A genetic material must carry out two jobs: duplicate itself and control the
development of the rest of the cell in a specific way.”-Francis Crick
Watson & Crick in action
Models for DNA replication1) Semiconservative model:
Daughter DNA molecules contain one parental strand and one newly
replicated strand.
2) Conservative model:
Parent strands transfer information to an intermediate, then the
intermediate gets copied. The parent helix is conserved, the daughter
helix is completely new.
3) Dispersive model:
Parent helix is broken into fragments, dispersed, copied then
assembled into two new helices. New and old DNA are completely
Dispersed.
(a) Hypothesis 1:
Semi-conservative replication
(b) Hypothesis 2:Conservative replication
Intermediate molecule
(c) Hypothesis 3:Dispersive replication
MODELS OF DNA REPLICATION
Meselson and Stahl Semi-conservative replication of DNA
The families of nitrogenous bases
DNA Replication
• Since DNA replication is semiconservative, therefore the helix must be unwound.
• John Cairns (1963) showed that initial unwinding is localized to a region of the bacterial circular genome, called an “origin” or “ori” for short.
• DNA replication is semiconservative. Each strand of both replication forks is being copied.
• DNA replication is bidirectional. Bidirectional replication involves two replication forks, which move in opposite directions
Evidence points to bidirectional replication
Label at both replication forks
The Enzymes of Replication
A 3’ hydroxylgroup is necessaryfor addition of nucleotides
DNA Polymerase contains a Proofreading subunit
Accuracy of DNA polymerases is essential.
-Error rate is less than 1 in 108
Proofreading by DNA polymerase
DNA Exonuclease
DNA replication is semi-discontinuous
Continuous synthesis
Discontinuous synthesis
Proteins & the Replication Fork
Protein complexes of the replication fork:
DNA polymerase
DNA primase
DNA Helicase
ssDNA binding protein
Sliding Clamp
Clamp Loader
DNA Ligase
DNA 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 duplex ahead of DNA polymerase creating single stranded DNA that can be used as 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 DNA easily). A “slidingclamp” 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
The supercoiling ahead of the fork needs to be relieved or tension wouldbuild up (like coiling as spring) and block fork progression.
Type I topoisomerases:Make nicks in one DNA strandsCan relieve supercoiling
Type II topoisomersases:Make 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
Supercoiling is relieved by the action of Topoisomerases
Topoisomerase I Action
Topoisomerase II Action
Topoisomerase II Action
Because dividing cells require greater topoisomerase activitydue to increased DNA synthesis, topoisomerase inhibitors are used 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-specific toposisomerase DNA gyrase
e.g. ciprofloxacin
Topoisomerases as drug targets
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 replicaton initiates the formation of bidirectionalreplication forks
Errors lead to over replication 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.
Origins of replication are strictly controlled so that they “fire” only once per cell cycle
Errors of DNA Replication and Disease
The rate of misincorporation of bases by DNA polymerase is extremely 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 sequence can have severe effects on the expression of a gene or the function of a protein.
Several inherited diseases are associated with expansion of trinucleotide repeat sequences.
Have no fear…it’s efficient!! Check out these stats…
Polymerase III
It’s fast: up to 1,000 dNTPs added/sec/enzyme
It’s highly processive: >500,000 dNTPs added before
dissociating
It’s accurate: makes 1 error in 107 dNTPs added, with
proofreading, this gives a final error rate of 1 in 1010 overall.