DNA polymerase mechanism

Figure 27.12. DNA Polymerase Mechanism. Two metal ions (typically, Mg2+) participate in the DNA polymerase reaction. One metal ion coordinates the 3 -hydroxyl group of the primer, whereas the phosphate group of the nucleoside triphosphate bridges between the two metal ions. The hydroxyl group of the primer attacks the phosphate group to form a new 0-P bond.

Modern Methods Used in DNA Polymerase Mechanism Studies 355... 

Even though this chapter has largely focused on the mechanism of correct dNTP incorporation and the molecular origin of fidelity, the computational approaches described here can also be utilized to study the effects of modified substrates in the active site of polymerases,as well as the effect of interacting proteins on catalysis. Overall, computational methods will continue to be a valuable tool for DNA polymerase mechanism studies. A complete understanding of polymerase mechanism and fidelity will surely be facilitated by future advancements in computational methodology, as well as the availability of new complex structures. 

The antiviral mechanism of action of acyclovir has been reviewed (72). Acyclovir is converted to the monophosphate in herpes vims-infected cells (but only to a limited extent in uninfected cells) by viral-induced thymidine kinase. It is then further phosphorylated by host cell guanosine monophosphate (GMP) kinase to acyclovir diphosphate [66341 -17-1], which in turn is phosphorylated to the triphosphate by unidentified cellular en2ymes. Acyclovir triphosphate [66341 -18-2] inhibits HSV-1 viral DNA polymerase but not cellular DNA polymerase. As a result, acyclovir is 300 to 3000 times more toxic to herpes vimses in an HSV-infected cell than to the cell itself. Studies have shown that a once-daily dose of acyclovir is effective in prevention of recurrent HSV-2 genital herpes (1). HCMV, on the other hand, is relatively uninhibited by acyclovir. 

The mode of action of PMEA may be quite similar to the mechanism by which (3)-HPMPA accomplishes its selective inhibitory activity against herpes vimses. Eor PMEA to reach its active triphosphate form, it needs only two phosphorylation steps. The triphosphate derivative of PMEA has a much stronger affinity for HIV-1 reverse transcriptase than for cellular DNA polymerases (175). Whether it is actually incorporated into DNA and terminates the growing DNA chain is currentiy under investigation. 

Each strand of the double helix is replicated simultaneously but by somewhat different mechanisms. A complex of proteins, including DNA polymerase, replicates the leading strand continuously in the 5 to 3 direction. The lagging strand is replicated discon-tinuously, in short pieces of 150-250 nucleotides, in the 3 to 5 direction. 

However, not all effects of NRTls on mitochondria can be explained by the DNA polymerase y hypothesis. Other mechanisms, either secondary to or independent of inhibition of DNA polymerase y are involved in NRTI toxicity (Moyle 2000a, 2000b Lewis et al. 2003). AZT is a potent inhibitor of mitochondrial DNA polymerase y but does not cause neuropathy in HIV patients (Dalakas 2001). Keswani and colleagues showed that NRTls caused direct mitochondrial toxicity through... 

Feig, D.I. and Loeb, L.A. (1993). Mechanisms of mutation by oxidative DNA damage reduced fidelity of mammalian DNA polymerase jS. Biochemistry 32, 4466—4473. 

Figure 7. Reaction mechanism of the 3, 5 -exonuclease subunit DNA polymerase I.

There are three mechanistic possibilities for catalysis by two-metal ion sites (Fig. 10). The first of these is the classic two-metal ion catalysis in which one metal plays the dominant role in activating the substrate toward nucleophilic attack, while the other metal ion furnishes the bound hydroxide as the nucleophile (Fig. 10 a). Upon substrate binding, the previously bridged hydroxide shifts to coordinate predominately with one metal ion. Enzymes believed to function through such a mechanism include a purple acid phosphatase [79], DNA polymerase I [80], inositol monophosphatase [81],fructose-1,6-bisphosphatase [82], Bam HI [83], and ribozymes [63]. 

Figure 10.14 The proposed transition state in the mechanism of the 3 -5 exonuclease activity of DNA polymerase I. (From Steitz and Steitz, 1993. Copyright (1993) National Academy of Sciences, USA.)...

Figure 21.6 One mechanism of activation of the cell cycle by a growth factor. Binding of growth factor to its receptor activates membrane-bound phospholipase-C. This hydrolyses phosphati-dylinositol bisphosphate in the membrane to produce the messengers, inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 results in release of Ca from an intracellular store. The increased Ca + ion concentration activates protein kinases including protein kinase-C (PK-C). DAG remains membrane-bound and also activates protein kinase-C (PK-C) which remains in the activated form as it travels through the cell where it phosphory-lates and activates transcription factors. This results in activation of genes that express enzymes involved in nucleotide synthesis, DNA polymerases and cyclins, which are all reguired for the cell cycle (See Chapter 20 for provision of nucleotides and cyclins for the cell cycle).

Telomeres are seqnences of six-nucleotide repeats found at the ends of the chromosomal DNA strands. Many thon-sands of repeat nnits (TTAGGG) may be present at the end of the 3 strand and (AATCCC) at the end of the 5 strand. These are present at the ends of the strands to overcome a problem posed by the semi-conservative mechanism of DNA replication, known as the end replication problem . Replication of the ends of the chromosomes presents par-ticnlar difficnlties, since DNA polymerase can only elon-... 

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