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Molecular and Cellular Biology, October 2003, p. 7152-7162, Vol. 23, No. 20
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.20.7152-7162.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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

Ariane Gratias and Mireille Bétermier*

Laboratoire de Génétique Moléculaire, CNRS UMR 8541, Ecole Normale Supérieure, 75005 Paris, France

Received 17 April 2003/ Returned for modification 26 May 2003/ Accepted 21 July 2003

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.

* Corresponding author. Mailing address: Laboratoire de Génétique Moléculaire, CNRS UMR 8541, Ecole Normale Supérieure, 46 rue d'Ulm, 75005 Paris, France. Phone: (0)1 44 32 39 47. Fax: (0)1 44 32 39 41. E-mail: betermie{at}wotan.ens.fr.

Molecular and Cellular Biology, October 2003, p. 7152-7162, Vol. 23, No. 20
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.20.7152-7162.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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