Meiosis, a specialized cell division involving a single cycle of DNA replication followed by two cycles of chromosome segregation, is a highly coordinated event. Chromosome segregation errors during meiosis have hazardous consequences such as birth defects, genetic diseases, infertility and malignancy. DNA Topoisomerase II (TOP-2) is required to relieve the topological stress associated with the unwinding of DNA during replication, recombination and sister chromatid segregation. In addition, TOP-2 has been also found to maintain chromosome structure. The role of TOP-2 has been studied well in mitosis but is not very well defined in meiosis. However, our lab recently characterized an allele of
top-2 [
top-2(
it7)] in C. elegans with a male meiosis-specific phenotype.
top-2(
it7) is a temperature sensitive (ts) allele and
top-2(
it7ts) sperm that develop at the restrictive temperature of 24°C have chromosome segregation defects at anaphase I of meiosis, which results in embryonic lethality after fertilization. In a genetic suppressor screen on
top-2(
it7ts animals, we identified 11 suppressors of
top-2(
it7) induced embryonic lethality. Interestingly, seven of the 11 suppressors identified had different point mutations in the same gene 5'-tyrosyl-DNA phosphodiesterase two (Tdp2, C. elegans
tdpt-1). Tdp2 is involved in the removal of trapped Top-2-DNA complexes. Currently, our lab is focused on elucidating the mechanism of
tdpt-1 mutant-mediated suppression of
top-2(
it7ts) embryonic lethality. Our data suggest that the
tdpt-1 suppressing mutations rescue the embryonic lethality of
top-2(
it7ts) to near wild-type levels, ameliorate the chromosome segregation defects of
top-2(
it7ts) worms and restore TOP-2 protein level to various degrees. We have validated and confirmed
tdpt-1 suppressing mutation to be a loss of function mutation using RNAi and CRISPR/Cas9. Similarly, we have found that
tdpt-1 mutations do not have a phenotype independently of
top-2(
it7) in meiosis. To unravel the molecular mechanism of TDPT-1 in the context of TOP-2 mutation for ensuring proper chromosome segregation during male gamete formation, we are currently examining if TDPT-1 is required to process TOP-2 induced DNA breaks during meiosis. Further, we plan to examine biochemical properties of TDPT-1, perform TDPT-1 expression and localization studies in relation to TOP-2 in the male germ line, and investigate TOP-2 as a potential binding partner of TDPT-1 during meiosis.