Chromosome Function and Replication - Basis for the Selective Inhibition of Chromosome Replication and Function

CHROMOSOME  FUNCTION  AND  REPLICATION

BASIS FOR THE SELECTIVE INHIBITION OF CHROMOSOME REPLICATION AND FUNCTION

As with protein synthesis, the mechanisms of chromosome replication and function are essentially the same in prokaryotes and eukaryotes. There are, however, important differences in the detailed functioning and properties of the enzymes involved and these differences are exploited by a number of agents as the basis of selective inhibition. The microbial chromosome is large in comparison with the cell that contains it (approximately 500 times the length of E. coli). It is therefore wound into a compact, supercoiled form inside the cell. During replication the circular double helix must be unwound to allow the DNA polymerase enzymes to synthesize new complementary strands. The shape of the chromosome is manipulated by the cell by the formation of regions of supercoiling. Positive supercoiling (coiling in the same sense as the turns of the double helix) makes the chromosome more compact. Negative supercoiling (generated by twisting the chromosome in the opposite sense to the helix) produces localized strand separation which is required both for replication and transcription. In a bacterium such as E. coli four different topoisomerase enzymes are responsible for maintaining the shape of DNA during cell division. They act by cutting one or both of the DNA strands; they remove and generate supercoiling, then reseal the strands. Their activity is essential for the microbial cell to relieve the complex tangling of the chromosome (both knotting and chain link formation) which results from progression of the replication fork around the circular chromosome. Type I topoisomerases 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. In E. coli topoisomerases I and III are both type I enzymes while topoisomerases II and IV are type II enzymes. Topoisomerase II (also known as DNA gyrase) and topoisomerase IV are essential enzymes which are inhibited by the fluoroquinolone group of antimicrobials. Topoisomerase II is responsible for introducing negative supercoils into DNA and for relieving torsional stress, which accumulates ahead of sites of transcription and replication. Topoiosomerase IV provides a potent decatenating (unlinking) activity that removes links and knots generated behind the replication fork.

The basic sequence of events for microbial chromosome replication is described below.

a)    Synthesis Of Precursors

Purines, pyrimidines and their nucleosides and nucleoside triphosphates are synthesized in the cytoplasm. At this stage the antifolate drugs (sulphonamides and dihydrofolate reductase inhibitors) act by interfering with the synthesis and recycling of the cofactor dihydrofolic acid (DHF). Thymidylic acid (2-deoxythymidine monophosphate, dTMP) is an essential nucleotide precursor of DNA synthesis. It is produced by the enzyme thymidylate synthetase by transfer of a methyl group from tetrahydrofolic acid (THF) to the uracil base on uridylic acid (2-deoxyuridine monophosphate, dUMP) (Figure 12.5). THF is converted to DHF in this process and must be reverted to THF by the enzyme dihydrofolate reductase (DHFR) before the cycle can be repeated. By inhibiting DHFR, the antifolates effectively block the production of dTMP and hence DNA synthesis.

The antifungal agent 5-fluorocytosine also interferes with these early stages of DNA synthesis. Through conversion to the nucleoside triphosphate it subsequently blocks thymidylic acid production through inhibition of the enzyme thymidylate synthetase (Figure 12.6).

The antiviral nucleosides aciclovir and ganciclovir are also converted to their respective nucleoside triphosphates in the cytoplasm of infected cells. They proceed to inhibit viral DNA replication either by inhibition of the DNA polymerase or by incorporation into DNA with subsequent termination of chain extension. Finally, the anti-HIV drug AZT acts in an analogous manner, being converted to the corresponding triphosphate and inhibiting viral RNA synthesis by the HIV reverse transcriptase.

b)    Unwinding Of The Chromosome

As described in section 4.1, the DNA double helix must unwind to allow access of the polymerase enzymes to produce two new strands of DNA. This is facilitated by topoisomerase II (DNA gyrase) which is the target of the fluoroquinolones. Some agents interfere with the unwinding of the chromosome by physical obstruction. These include the acridine dyes, of which the topical antiseptic proflavine is the most familiar, and the antimalarial acridine mepacrine. They prevent strand separation by insertion (intercalation) between base pairs from each strand, but exhibit very poor selective toxicity.

c)    Replication Of DNA Strands

The unwound DNA strands are kept unfolded during replication by binding a protein called Albert’s protein. A series of enzymes produce new strands of DNA using each of the separated strands as templates. One strand is produced continuously. The other is produced in a series of short strands called Okazaki fragments that are joined by a DNA ligase. The entire process is carefully regulated, with proofreading stages to check that each nucleotide is correctly incorporated as specified by the template sequence. There are no therapeutic agents yet known which interfere directly with the DNA polymerases.

d)   Transcription

The process of transcription, the copying of a single strand of mRNA sequence using one strand of the chromosome as a template, is carried out by RNA polymerase. This is a complex of four proteins (2 α, 1 β and 1 β′ subunits) which make up the core enzyme. Another small protein, the σ factor, joins the core enzyme, which binds to the promoter region of the DNA preceding the gene that is to be transcribed. The correct positioning and orientation of the polymerase is obtained by recognition of specific marker sites on the DNA at positions −10 and −35 nucleotide bases before the initiation site for transcription. The σ factor is responsible for recognition of the initiation signal for transcription and the core enzyme possesses the activity to join the nucleotides in the sequence specified by the gene. Mammalian genes possess an analogous RNA polymerase but there are sufficient differences in structure to permit selective inhibition of the microbial enzyme by the semisynthetic rifamycin antibiotics rifampicin and rifabutin.