Processing of Double-Strand Breaks Is Involved in the Precise Excision of Paramecium Internal Eliminated Sequences

Abstract

In ciliates, the development of the somatic macronucleus involves the programmed excision of thousands of internal eliminated sequences (IES) scattered throughout the germ line genome. Previous work with Tetrahymena thermophila has suggested that excision is initiated by a staggered double-strand break (DSB) at one IES end. Nucleophilic attack of the other end by the 3′OH group carried by the firstly broken chromosome end leads to macronuclear junction closure. In this study, we mapped the 3′OH and 5′PO₄ groups that are developmentally released at Paramecium IES boundaries, which are marked by two conserved TA dinucleotides, one of which remains in the macronuclear genome after excision. We show that initiating DSBs at both ends generate 4-base 5′ overhangs centered on the TA. Based on the observed processing of the 5′-terminal residue of each overhang, we present a new model for the precise closure of macronuclear chromosomes in Paramecium tetraurelia, different from that previously proposed for Tetrahymena. In our model, macronucleus-destined broken ends are aligned through the partial pairing of their 5′-nTAn-3′ extensions and joined after trimming of the 5′ flaps.

ACKNOWLEDGMENTS

We thank Eric Meyer for his constant support throughout the course of this work. We are extremely grateful to Jim Forney and Erika Snodderley for communicating the sequence of IES 51A-4814 prior to publication. We thank Jérôme Chal and Carole Escartin for their participation in the optimization of LMPCR assays, and we thank Edouard Bray for his help with the design of our Web page. Many thanks to Tom Doak and all members of E. Meyer's laboratory for stimulating discussions and to Sandra Duharcourt, Anne Le Mouël, Sophie Malinsky, E. Meyer, and Linda Sperling for critical reading of the manuscript.

This work was supported by the Association pour la Recherche sur le Cancer (grant 5733), the Centre National de la Recherche Scientifique (Soutien aux jeunes équipes), and the Comité de Paris de la Ligue Nationale contre le Cancer (grant 75/01-RS/73). A. Gratias was the recipient of a doctoral fellowship from the French Ministère de la Recherche and is currently supported by the Association pour la Recherche sur le Cancer.