Ultrastructural characterization of RPA-containing domains in nuclei assembled in Xenopus egg extracts

We describe novel structural domains in in vitro reconstituted Xenopus sper m nuclei, which we term RPA bodies; RPA is the only known marker of these s tructures. These bodies contain DNA and represent special chromatin domains as seen by transmission electron microscopy, We show that RPA bodies exhib it a similar ultrastructure in nuclei assembled in high-speed supernatant ( HSS) of Xenopus egg extract and in nuclei assembled in HSS supplemented wit h low-speed supernatant (HSS + LSS nuclei). Moreover, RPA bodies are also f ormed when sperm chromatin containing double-stranded DNA breaks is incubat ed with HSS of egg extracts. RPA bodies appear to be compartmentalized. By immunoelectron microscopy we show that RPA is preferentially localized at t he periphery of the bodies where DNA synthesis also occurs in HSS + LSS nuc lei. (C) 2000 Academic Press.