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Organization of DNA
Supercoiling
mt DNA and the DNA of most prokaryotes are
closed circular structures. These molecules may
exist as relaxed circles or as supercoiled structures
in which the helix is twisted around itself in threedimensional space.
Supercoiling results from strain on the molecule
caused by under- or overwinding the double helix:
Chromosome structure
(Nucleofilament)
Electron micrograph of a
protein-depleted human
metaphase chromosome,
showing the residual
chromosome
scaffold and loops of DNA.
Individual DNA fibers can be
best seen at the edge of the
DNA loops. Bar = 2. (From
Paulson JR,
Laemmli UK [1977] The
structure of histone-depleted
metaphase chromosomes. Cell
12:817828.
An Interphase Nucleus
The bacterial chromosome is a closed, doublestranded circular DNA molecule having a single
origin of replication.
Separation of the two parental strands of DNA
creates two replication forks that move away from
each other in opposite directions
Replication is, thus, a bidirectional process.
The two replication forks eventually meet,
resulting in the production of two identical
circular molecules of DNA.
3'5'-Exonuclease activity enables DNA polymerase III to proofread the newly synthesized
DNA strand.
*Certain DNA and RNA polymerases require RNA templates. These enzymes are
most commonly associated with viruses.
Similarities include:
The newly synthesized strand is made in the 5'3'
direction.
The template strand is scanned in the 3'5' direction.
The newly synthesized strand is complementary and
antiparallel to the template strand.
Each new nucleotide is added when the 3' hydroxyl
group of the growing strand reacts with a nucleoside
triphosphate, which is base-paired with the template
strand. Pyrophosphate (PPi, the last two phosphates)
is released during this reaction.
Differences include:
The substrates for DNA synthesis are the dNTPs, whereas the
substrates for RNA synthesis are the NTPs.
DNA contains thymine, whereas RNA contains uracil.
DNA polymerases require a primer, whereas RNA
polymerases do not. That is, DNA polymerases cannot
initiate strand synthesis, whereas RNA polymerases can.
DNA polymerases can correct mistakes ("proofreading"),
whereas RNA polymerases cannot. DNA polymerases have
3'5' exonuclease activity for proofreading.
Discontinuous
synthesis of DNA.
Removal of RNA primer and filling of the resulting gaps by DNA polymerase-I
Formation of a phosphodiester
bond by DNA ligase.
Telomerase
Telomeres are repetitive sequences at the ends of
linear DNA molecules in eukaryotic chromosomes.
With each round of replication in most normal
cells, the telomeres are shortened because DNA
polymerase cannot complete synthesis of the 5'
end of each strand.
This contributes to the aging of cells, because
eventually the telomeres become so short that the
chromosomes cannot function properly and the
cells die.
Reverse Transcriptase
Reverse transcriptase is an RNA-dependent DNA
polymerase that requires an RNA template to
direct the synthesis of new DNA. Retroviruses,
most notably HIV, use this enzyme to replicate
their RNA genomes.
DNA synthesis by reverse transcriptase in
retroviruses can be inhibited by AZT, ddC, and
ddI.
Bridge to Pharmacology
Quinolones and DNA Gyrase: Quinolones and
fluoroquinolones inhibit DNA gyrase (prokaryotic
topoisomerase II), preventing DNA replication and
transcription. These drugs, which are most active against
aerobic gram-negative bacteria include:
Nalidixic acid
Ciprofloxacin
Norfloxacin
Resistance to the drugs has developed overtime; current
uses include treatment of gonorrhea, and upper and lower
urinary tract infections in both sexes.
DNA REPAIR
The structure of DNA can be damaged in a number of
ways through exposure to chemicals or radiation.
Incorrect bases can also be incorporated during
replication.
Multiple repair systems have evolved, allowing cells to
maintain the sequence stability of their genomes.
If cells are allowed to replicate their DNA using a
damaged template, there is a high risk of introducing
stable mutations into the new DNA.
Thus any defect in DNA repair carries an increased risk
of cancer.
Most DNA repair occurs in the G1 phase of the
eukaryotic cell cycle. Mismatch repair occurs in the G2
phase to correct replication errors.
Homologous Recombination
Here the broken ends are repaired using the information
on the intact sister chromatid (available in G2 after chromosome
duplication), or on the
homologous chromosome (in G1; that is, before each chromosome
has been duplicated). This requires searching around in the nucleus
for the homolog a task sufficiently uncertain that G1 cells usually
prefer to mend their DSBs by NHEJ, or on
the same chromosome if there are duplicate copies of the gene on the
chromosome oriented in opposite directions (head-to-head or back-toback).
Non-cross over
recombinant
Cross over
recombinant
Key differences:
- Exonuclease
- DNA polymerase