Chromosome function and replication

During replication the circular double helix must be unwound to allow the DNA
polymerase enzymes to synthesize new complementary strands.

In a bacterium such E. coli for different topo-isomerase enzymes are responsible for
maintaining the shape of DNA during cell division. They act by cutting one or both of
the DNA strands.

Type I topo-isomerases: cut one strand of DNA and pass the other strand through the
gap before resealing.
Type II: enzymes cut both strands and pass another double helical section of the DNA
through the gap before resealing.

E. coli topo-isomerases I and III are both type I enzymes

Topo-isomerase II and IV are type II enzyme and considered essential.
Topo-isomerase II- responsible for introducing negative supercoils into DNA.
Topo-isomerase IV- provides a potent unlinking activity.

Synthesis of precursor

Purines, pyrimidines and their nucleosides and nucleoside triphosphates are

synthesized in the cytoplasm.
Thymidylic acid- essential nucleotide precursor of DNA synthesis.
Antifungal agent 5-fluorocytosine also interferes with these early stages of DNA
synthesis.

Unwinding of the chromosome

The DNA double helix must unwind to allow access of the polymerase enzymes to
produce two new strands of DNA.
Some agents interfere with the unwinding of the chromosome by physical
obstruction.

Replication of DNA strands

Unwound DNA strands are kept unfolded during replication by binding a protein
called Alberts protein.
One strand is produced continuously while the other is produced in a series of short
strands called Okazaki fragments.
The entire process is carefully regulated with proofreading stages to check that each
nucleotide is correctly incorporated.

Transcription

Copying of a single strand of mRNA sequence using one strand of the chromosome

as a template, is carried out by RNA polymerase.

Fluoroquinolones

Selectively inhibit topo-isomerases II and IV.

Nitroimidazoles (metronidazole, tinidazole) and nitrofurans (nitrofiratoin)

These agents cause DNA strand breakage.

The nitro group of metronidazole is converted to a nitronate radical by the low redox
potential within cells.
Pyruvate ferredoxin oxidoreductase- major metabolic pathway in anaerobic
bacteria and protozoa.

Rifampicin and rifabutin

Rifampicin- acts on the B-subunit of RNA polymerase. Binding of just one molecule
of it will inhibit initiation stage of transcription.
The action of rifampicin is specific for the microbial RNA polymerase.
Rifabutin- enhanced activity against Mycobacterium avium complex.

5-Fluorocytosine

This antifungal agent inhibits DNA synthesis at the early stages involving production
of the nucleotide thymidylic acid.
5-FC is an important antifungal agent in the treatment of life-threatening infections,
resistance can occur due to active efflux of the drug from the cells before it can
inhibit DNA synthesis.

Folate antagonists

Folic acid- important cofactor in all living cells

Tetrahydrofolate (THF)- carrier of a single-carbon fragments which are used in the
synthesis of adenine, guanine, thymine, and methionine.
Thymidylate synthetase- important folate-dependent enzyme which produces
TMP.
Dihydrofolate reductase- enzyme responsible for the reduction of DHF to THF.
DHFR- double enzyme which also contains the thymidylate synthetase activity.

Sulphonamides

Competitively inhibit the incorporation of PABA into dihydropteroic acid.

DHFR inhibitors

Trimethoprim- selective inhibitor of bacterial DHFR.

Pyrimethamine- selective inhibitor of plasmodial DHFR.
DHFR inhibitors- used in combination with sulphonamide to achieve a double
interference with folate metabolism.

Cytoplasmic membrane

The integrity of cytoplasmic membrane is vital for the normal functioning of all cells.
The stability of all membranes is maintained by a combination of non-covalent
interactions between the constituents involving ionic, hydrophobic, and hydrogen
bonding.

Polymyxins

Polymyxin E (colistin)- used in the treatment of serious gram negative bacterial

infections.
It binds tightly to the lipid A component of LPS in the outer membrane of gram
negative bacteria.
Polymyxin molecules can then penetrate to the cytoplasmic membrane where they
can bind to phospholipids, disrupt membrane integrity, and cause irreversible
leakage of cytoplasmic membrane.

Daptomycin

The acyl tail portion of the compound inserts itself into the cytoplasmic membrane
and drug molecules aggregate together forming channels.
The leakage of potassium ions from the cells results in inhibition of
macromolecular synthesis and cell death.

Polyenes

Amphotericin B and nystatin- most commonly used antifungal agents. They derive
their action from their strong affinity towards sterols, particularly ergosterol.
Kidney damage is a major problem where polyenes are used systemically to treat
severe fungal infections.
The problem can be reduced but not eliminated by administration of amphotericin as
a lipid complex or liposome.

Imidazoles and triazoles

The azole antifungal drugs act by inhibiting the synthesis of the sterol components
of the fungal membrane.
Imidazoles and triazoles cause repid defects in fungal membrane integrity due to
reduced level of ergosterol.

Terbinafine

synthetic antifungal agent that inhibits teh enzyme squalene epoxidase at an early
stage in fungal sterol biosynthesis.