Summary Piwi-interacting RNAs (piRNAs) silence transposons in the germ line of animals. They are thought to derive from long primary transcripts spanning transposon-rich genomic loci, “piRNA clusters.” piRNAs are proposed to direct an auto-amplification loop in which an antisense piRNA, bound to Aubergine or Piwi protein, directs the cleavage of sense RNA, triggering production of a sense piRNA bound to the PIWI protein Argonaute3 (Ago3). In turn, the new piRNA is envisioned to direct cleavage of a cluster transcript, initiating production of a second antisense piRNA. Here, we describe strong loss-of-function mutations in ago3, allowing a direct genetic test of this model. We find that Ago3 acts to amplify piRNA pools and to enforce on them an antisense bias, increasing the number of piRNAs that can act to silence transposons. We also detect a second piRNA pathway centered on Piwi and functioning without benefit of Ago3-catalyzed amplification. Transposons targeted by this second pathway often reside in the flamenco locus, which is expressed in somatic ovarian follicle cells, suggesting a role for piRNAs beyond the germ line.

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Time resolved data of DNA damage and repair after radiotherapy elucidates the relation between damage, repair, and cell survival. While well characterized in vitro, little is known about the time-course of DNA damage response in tumors sampled from individual patients. Kinetics of DNA damage after radiotherapy was assessed in eight dogs using repeated in vivo samples of tumor and co-irradiated normal tissue analyzed with comet assay and phosphorylated H2AX (γH2AX) immunohistochemistry. In vivo results were then compared (in silico) with a dynamic mathematical model for DNA damage formation and repair. Maximum %DNA in tail was observed at 15–60 min after irradiation, with a rapid decrease. Time-courses of γH2AX-foci paralleled these findings with a small time delay and were not influenced by covariates. The evolutionary parameter search based on %DNA in tail revealed a good fit of the DNA repair model to in vivo data for pooled sarcoma time-courses, but fits for individual sarcoma time-courses suffer from the heterogeneous nature of the in vivo data. It was possible to follow dynamics of comet tail intensity and γH2AX-foci during a course of radiation using a minimally invasive approach. DNA repair can be quantitatively investigated as time-courses of individual patients by integrating this resulting data into a dynamic mathematical model.

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