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.