Drosophila TDP1 Ortholog Important for Longevity and Nervous System Maintenance

Rough eye phenotype of TDP1 knockout flies treated with topoisomerase I (Top1) inhibitors or bleomycin. (A) Representative scanning electron microscopy (SEM) image of a normal eye from an untreated tdp1-knockout flies. (B) SEM image of the eye of a fly treated with camptothecin or the non-camptothecin Top1 inhibitor (LMP776) in phase I clinical trial.

Rough eye phenotype of TDP1 knockout flies treated with topoisomerase I (Top1) inhibitors or bleomycin. (A) Representative scanning electron microscopy (SEM) image of a normal eye from an untreated tdp1-knockout flies. (B) SEM image of the eye of a fly treated with camptothecin or the non-camptothecin Top1 inhibitor (LMP776) in phase I clinical trial.

As the molecule responsible for encoding a cell’s hereditary information, DNA must maintain its integrity. However, nucleic acids are vulnerable to damage by a number of endogenous and exogenous insults, such as reactive oxygen species or enzymes that react with DNA. Thus, other enzymes are tasked with repairing damaged DNA, including tyrosyl-DNA phosphodiesterase 1 (TDP1), which frees the 3’ ends of DNA that are blocked by proteins and oxidized bases to allow the ligation of strand breaks. Yeast, mice, and humans that express mutants of TDP1 have a reduced capacity to repair oxidative or topoisomerase-induced damage. A Drosophila TDP1 ortholog, glaikit (gkt), has been reported, but its function in DNA repair has not been evaluated because, surprisingly, gkt knockout flies were not viable.

To begin investigating the role of gkt, Yves Pommier, M.D., Ph.D., of CCR’s Developmental Therapeutics Branch, Howard Nash, M.D., Ph.D., (deceased) of the National Institute of Mental Health, and their colleagues decided to study mutant flies with a disruptive insertion, c03958, located 38 nucleotides upstream of the gkt gene. In contrast to the previous report but in agreement with data from mice, the mutant flies were healthy and fertile and provided a useful model system.

The researchers first tested whether TDP1 activity was impaired in c03958-containing flies. They combined a synthetic nucleopeptide that mimics a topoisomerase I-DNA complex normally repaired by TDP1 with varying amounts of protein extracts from wild type or c03958 flies. While an 81-fold dilution of the extract from wild type flies continued to show significant phosphodiesterase activity, even the most concentrated c03958 fly extract demonstrated almost none, and the weak activity observed was likely from other, less effective and less specific enzymes. The investigators next examined the distribution of TDP1 expression in flies by generating an antibody to a peptide in its C terminus. In wild type flies, they observed high TDP1 protein levels in the head and weaker levels in the body, consistent with the reported distribution of TDP1 mRNA. In contrast, c03958 flies had no detectable TDP1 protein, indicating that the insertion does disrupt expression of the enzyme. These results demonstrate that the gtk gene does encode the Drosophila TDP1 protein.

Because the types of damages recognized by TDP1 can lead to increased mortality if not repaired, the scientists monitored the lifespans of wild type and c03958 flies. Female, but not male, c03958 flies lived significantly fewer days than their wild type counterparts. The mutant female flies also exhibited impaired climbing ability at older ages. Both phenotypes could be reversed with re-expression of TDP1 in the brain, demonstrating the important housekeeping role of the enzyme for the nervous system.

Disruption in DNA repair can also lead to defects in Drosophila eye development, manifesting as a rough eye. Without any treatment, the researchers observed that the eyes of the c03958 flies were mostly normal. However, feeding larvae camptothecin, a topoisomerase I inhibitor, led to rough eyes in both wild type and c03958 flies, but the phenotype was more severe in the mutants. The investigators saw similar results with larvae fed either of two other DNA-damaging agents that depend on TDP1 for repair, bleomycin and a novel topoisomerase I inhibitor in clinical development at the NCI, LMP776. In contrast, they found no rough eye phenotype in adult c03958 flies fed etoposide, a topoisomerase II inhibitor. Re-expressing TDP1 in the neurons of c03958 flies partially restored the fraction of rough-eye adults fed camptothecin or bleomycin as larvae but not to the level of wild type controls. Because of the complexity of Drosophila eye development, the incomplete correction of the rough eye phenotype is likely due to loss of TDP1 activity in cells other than neurons.

Together, these studies demonstrate that the TDP1 activity in Drosophila is encoded by the gkt gene. They also show that the ability of TDP1 to repair damaged DNA is essential for normal longevity and nervous system function in flies and may have important implications for higher organisms, including the role of TDP1 in the resistance of cancer cells to topoisomerase I inhibitors.

Summary Posted: 11/2014

Reference

Guo DY, Dexheimer TS, Pommier Y, and Nash HA. Neuroprotection and repair of 3’-blocking DNA ends by glaikit (gkt) encoding Drosophila tyrosyl-DNA phosphodiesterase 1 (TDP1). PNAS. October 20, 2014 PubMed Link