• Hamna Jameel, Medical Diagnostics, University of Delaware

• Aimee Jaramillo-Lambert, Biological Sciences, University of Delaware

Can mutations in the catalytic site of the TDPT-1 enzyme suppress top-2-induced embryonic lethality?  

Hamna Jameel, Carolyn Remsburg, and Aimee Jaramillo-Lambert

Department of Biological Sciences, University of Delaware, Newark DE 19716

The top-2 gene plays a crucial role in DNA replication and cell division. Mutations in this gene can lead to serious negative health outcomes including cancer and infertility. For example, in the model organism C. elegans the top-2(it7) mutation results in non-functional sperm leading to embryonic lethality. Previous studies from our lab have found that mutations in an enzyme, TDPT-1, can suppress mutations in the top-2 gene. The goal of this study is to determine whether loss of TDPT-1 enzymatic activity, rather than loss of structural support or protein interactions, can suppress top-2(it7) embryonic lethality. We employed a combination of polymerase chain reaction (PCR), gel electrophoresis, and embryonic viability assays to analyze mutations that specifically disrupt the catalytic domain of TDPT-1. The TDPT-1 catalytic site comprises four amino acids (N126, E158, D271, and H353). The mutant strains analyzed included a single mutation strain tdpt-1(syb8907), which changed H353 to A, and a triple mutation strain, tdpt-1(syb9110), which contains H353A, N126A, and E158A. Our results indicate that N2 (wild type) is 98% embryonically viable, tdpt-1(syb8907) is 97.3% viable, and tdpt-1(syb9110) is 96.3%  viable. tdpt-1(syb8907) and tdpt-1(syb9110) are statistically different from N2. However, there is no significant difference between the percent viability of tdpt-1(syb8907) and tdpt-1(syb9110). Future research will combine these tdpt-1 mutations with the top-2(it7) mutation to determine suppression. Understanding if mutations in an enzyme can suppress mutations in the top-2 gene can lead to an improved understanding of DNA replication and fertility.