Electronic Journal of Biotechnology

New molecular biologist perspective and insight: DNA topoisomerases production by recombinant DNA technology for medical laboratory application and pharmaceutical industry.

Electronic Journal of Biotechnology ISSN: 0717-3458	Vol. 16 No. 6, Issue of November 15, 2013
© 2013 by Pontificia Universidad Católica de Valparaíso -- Chile	Received March 28, 2013 / Accepted August 28, 2013
DOI: 10.2225/vol16-issue6-fulltext-6

Abstract

DNA topoisomerases are essential enzymes that control the topological state of DNA replication during mitosis. These enzymes are classified based on their mechanisms and physical properties. During mitosis, superhelical DNA must be unwound or relaxed by DNA topoisomerases prior to a decoding step by DNA processing enzymes, such as DNA polymerase and RNA polymerase. By blocking the reaction of resealing the breaks in the DNA ultimately can result in cellular death. Compounds that inhibit the catalytic function of these enzymes can serve as potential anticancer agents. DNA topoisomerases are found in nature and used as high quality and well-validated targets for the screening of potential anticancer agents. Our current work focuses on determining potential anticancer agents from natural resources using DNA topoisomerases as the screening targets. Large scale production of these enzymes using recombinant DNA technology in our academic laboratory is utilised to avoid dependence on expensive commercially available enzymes. The in-house produced enzymes can also be used to enhance our research in the field of molecular medicine by providing an enzyme source that can be used to screen potential anticancer agents, and for other newly developed diagnostic and medical research projects in the near future as well as a step in moving our efforts into the industrial sector.

Introduction

DNA topoisomerases are essential enzymes that control the topological state of DNA during cellular processes in both prokaryotic and eukaryotic cells. The cellular processes include DNA replication, transcription, recombination and chromatin segregation, to control the synthesis of proteins and to facilitate DNA replication during mitosis (Champoux, 2001; Durand-Dubief et al. 2011). The types of organisms used in the studies of DNA topoisomerases include Escherichia coli, Staphylococcus aureus, yeasts, plants from the genus Arabidopsis, flies from the genus Drosophila and human. Several viruses, such as bacteriophage T4 and animal vaccinia viruses, can also encode DNA topoisomerases. All organisms contain at least two classes of DNA topoisomerases, namely type I and type II DNA topoisomerases in which the enzymes are classified based on their mechanisms and physical properties (Champoux, 2001; Salerno et al. 2010). For example, E. coli has two type I DNA topoisomerases (DNA topoisomerase I and DNA topoisomerase III) and two type II DNA topoisomerases (DNA topoisomerase II or gyrase and DNA topoisomerase IV) (Kato et al. 1992; Peng and Marians, 1993). The DNA topoisomerase subtypes (A, B or C) are differentiated by amino acid sequences or structures (Champoux, 2001, Vos et al. 2011). For example, enzymes that cleave only one strand of DNA are defined as type I with a further classification of type I-A subtypes for proteins linked to a 5’-phosphate and type I-B subtypes for proteins attached to a 3’-phosphate during the relaxation of the cleavable DNA. The enzymes are further divided into subfamilies based on the structural changes induced by gene duplication, such as DNA topoisomerase IIα and IIβ (Wang, 1996, Champoux, 2001).

Other applications of DNA topoisomerases

DNA topoisomerases can also be used for other molecular biology laboratory applications. The DNA topoisomerase I from the vaccinia virus has been widely used to produce the DNA topoisomerase-activated adapter for various modifications and cloning of PCR products by inserting a DNA fragment into a plasmid without DNA ligase. This cloning method rapidly incorporates the T7 promoter into a plasmid for a PCR product with 3’ deoxyadenosine (A) overhangs, using a plasmid that has been engineered to have a linearized strand with 3’ deoxythymidine (T) overhangs. The DNA topoisomerase I from vaccinia virus covalently binds to the linearized plasmid at a specific site, cleaving the phosphodiester backbone after the 5’-CCCTT in a single strand of the plasmid DNA (Figure3). The phosphotyrosyl bond between the DNA and enzyme can be subsequently replaced by the 5’-hydroxyl of the original strand, releasing the enzyme after a religation with the external DNA, such as PCR product or DNA fragment (Shuman,1994). The PCR product, complement of the 3’-T overhang of the plasmid, is then allowed to quickly ligate with the DNA topoisomerase I in salt and without requiring a DNA ligase. This application has been developed by different manufacturers, including Invitrogen, to produce a number of plasmids for the effective cloning of a PCR product, providing a powerful and convenient tool for the rapid modification of a PCR product.

The perspectives of our current research

Current cancer drugs and treatment strategies have been reported to cause many adverse side effects to cancer patients. The side effects include hair loss, weakened immune system, vomiting, sickness etc. Improvements in effective cancer treatments have been limited despite the advances made in cancer research in the medical laboratory. Moreover, drugs used for chemotherapy can be excessively expensive (more than RM 1,000 per dose), reducing affordability for some patients. This restriction in patient access makes the search for effective and affordable anticancer agents from natural products more urgent and critical as these resources may be more affordable to cancer patients with low living wages. Natural products, such as garlic, ginseng, grape, shallot, green tea and onion, contain a high level of flavonoids that have been proven in vitro to have anticancer properties, including inhibitory effect on cancer cell growth via induction of apoptosis and autophagy (Singletary and Milner, 2008). For example, chrysin, which has been found to be the most effective of all the flavonoids tested, has equal potency to the drugs used for chemotherapy, such as tamoxifen. Indeed, natural products may be ideal resources for anticancer agents. Indeed, many natural products have yet to be scientifically proven to have anticancer effects using the above mentioned mechanisms. Therefore, large amounts of DNA topoisomerases are required to perform preliminary screening of potential anticancer agents from natural products. Large quantity of in-house produced DNA topoisomerases are also needed to develop standard, robust, rapid and sensitive preliminary screening assays for cancer diagnoses and predictions of the responses to chemotherapy, as well as for other molecular biology applications in our laboratory.

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