Processing of Double-Strand Breaks Is Involved in the Precise Excision ofParameciumInternal 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 3OH group carried by the firstly broken chromosome end leads to macronuclear junction closure. In this study, we mapped the 3OH and 5PO 4 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.Recombination processes involving the cutting and rejoining of DNA molecules have been found in a wide range of organisms, and two types of molecular mechanisms have been documented (reviewed in reference 5): conservative site-specific recombination, leading to the deletion/insertion or inversion of DNA sequences, involves the transient formation of covalent protein-DNA intermediates, while other reactions, such as the transposition of most class II transposons, generate DNA intermediates. Ciliates provide attractive models for the study of programmed DNA rearrangements and their regulation, because their entire genome is remodeled at each sexual cycle (reviewed in references 13 and 34

Analysis of sequence variability in the macronuclear DNA of Paramecium tetraurelia: A somatic view of the germline

“…IES excision is precise in Paramecium, as required for gene expression and as shown by studies of the mechanism (Gratias and Bétermier 2003). However, up until now nothing was known of the efficiency of the IES excision process.…”

“…The first DNA elimination pathway is responsible for the removal of some 60,000 short, unique-copy elements (IES, for Internal Eliminated Sequence) that interrupt both coding and non-coding sequences. IESs are bound by 5Ј-TA-3Ј dinucleotides and their perfectly precise excision is accomplished by a mechanism that produces double-stranded breaks followed by end joining (Gratias and Bétermier 2003). One TA dinucleotide remains in the chromosome after excision of the element.…”

Maternal noncoding transcripts antagonize the targeting of DNA elimination by scanRNAs in Paramecium tetraurelia

“…IESs all appear to be single-copy in the haploid genome and are always flanked by two 5Ј-TA-3Ј dinucleotides, one of which is retained at the macronuclear junction after excision. They do not share any other strictly conserved motifs, although a degenerate consensus at their extremities (Klobutcher and Herrick 1995) seems to be of importance for the staggered doublestrand breaks that initiate IES excision (Gratias and Betermier 2003). The molecular mechanisms underlying the specific recognition of such a large number of different germline sequences remain poorly understood.…”

Subtraction by addition: domesticated transposases in programmed DNA elimination: Figure 1.

“…1A; Gratias and Betermier 2003). The Betermier group (Gratias and Betermier 2003) had shown previously that IESs are removed by a mechanism that generates a 4-base staggered cut, leaving 59 overhangs; Tc transposases generate 2-base 39 overhangs (van Luenen et al 1994). Like the cleavage observed at IES ends, piggyBac transposases generate 4-base, 59 overhangs of the sequence TTAA (Mitra et al 2008).…”

“…For example, the consensus sequence flanking Paramecium IESs is reminiscent of Tc/mariner TIRs; however, the type of DSB characterized at the end of each IES did not match that expected for this family of elements (see Fig. 1A; Gratias and Betermier 2003). The Betermier group (Gratias and Betermier 2003) had shown previously that IESs are removed by a mechanism that generates a 4-base staggered cut, leaving 59 overhangs; Tc transposases generate 2-base 39 overhangs (van Luenen et al 1994).…”