Enzyme involved in synthesis and repair

Applied Molecular Evolution of Enzymes Involved in Synthesis and Repair of DNA... 

I is involved in synthesis, proofreading, repair, and removal of RNA primers a a polymerizing enzyme... 

Polymerase II (pol II) is mostly involved in proofreading and DNA repair. Polymerase I (pol I) completes chain synthesis between Okazaki fragments on the lagging strand. Eukaryotic cells have counterparts for each of these enzymes plus some additional ones. A comparison is shown in Table 36—6. 

Genetic recombination arises by exchange of homologous segments of DNA between viral genomes, most often during the replication process. The enzymes involved in recombination are DNA polymerases, endonucleases, and ligases, which also play a role in DNA repair and synthesis processes. 

The TS mediates the conversion of 2-deoxyuridine monophosphate (dUMP) into deoxythymidine monophosphate (dTMP). This enzymatic methylation reaction is a key step in the synthesis of DNA and involves a ternary complex between the substrate, the enzyme and the co-factor [methylene tetrahydrofolic acid (CH2FAH4)] (Fig. 24) [8,80,81], This transformation represents the sole de novo source of dTMP, a building block for DNA synthesis and repair [82]. 

In E. coli the synthesis of many enzymes involved in repair is regulated by the so-called SOS system. Two proteins, lexA and recA, form the working machinery of this regulatory system (fig. 26.19). Under normal conditions the lexA protein inhibits the expression of about 17 genes (the din genes), the encoded proteins of which are... 

Random oligonucleotide mutagenesis was first applied to promotor sequences that regulate the production of enzymes in cells [21] and was the first method used to alter systematically the functions of enzymes by directed evolution [22], Based on our experience, we will focus on this approach and emphasize recent applications of this methodology to enzymes involved in DNA repair and synthesis, including DNA polymerase enzymes, thymidylate synthase, thymidine kinase, and 06-alkylguanine-DNA alkyltransferase. 

Indirect evidence for DNA repair as a determinant in acquired resistance was obtained with the discovery that a mouse leukemia cell line 50-fold resistant to cis-DDP had a fourfold increase in the level of DNA polymerase p, an enzyme involved in DNA repair (73). This hypothesis was supported by a study in which a human ovarian carcinoma cell line 20-fold resistant to cis-DDP showed two to three times more repair synthesis than the parental cell line after a 1-h exposure to the drug (74). Also, studies in L1210 cells found that, although d(GpG), d(ApG), and d(ApNpG) crosslinks formed by [ H][Pt(en)Cl2] were all repaired, resistant cells repaired fourfold more adducts during an initial 6-h phase of repair (39). 

Five DNA polymerases have been found in E. coU. Polymersase III is primarily responsible for the synthesis of new strands. The first polymerase enzyme discovered, polymerase I, is involved in synthesis, proofreading, and repair. Polymerase II, IV, and V function as repair enzymes under unique conditions. 

Cd(II) (4pM) blocked UV-induced unscheduled DNA synthesis (i.e., repair replication) in human cells, and this inhibition was blocked by a higher molarity of Zn(II) (Nocentini 1987). The same concentration of Cd(II) caused the accumulation of DNA strand breaks, and this was also blocked by a tenfold molar excess of Zn(II) (Nocentini 1987). Because the initial decrease in size of DNA from UV-irradiated cells was not affected by Cd(II), it is unlikely that the initial incision steps of excision repair were inhibited. However, a more toxic treatment of cells with CdCl2 did inhibit pyrimidine dimer removal (Snyder et al. 1989). UV-induced cytotoxicity was increased by Cd(II), an effect also blocked by Zn(II). Taken together, these results suggest that Cd(II) and Zn(II) compete for a common binding site on enzymes involved in DNA replication and repair. Cd(II) inhibits repair of X-ray-induced strand breaks (DNA ligase ) (Snyder et al. 1989). 

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