Product analysis illuminates the final steps of IES deletion in Tetrahymena thermophila

Abstract

DNA sequences (IES elements) eliminated from the developing macronucleus in the ciliate Tetrahymena thermophila are released as linear fragments, which have now been detected and isolated. A PCR-mediated examination of fragment end structures reveals three types of strand scission events, reflecting three steps in the deletion process. New evidence is provided for two steps proposed previously: an initiating double-stranded cleavage, and strand transfer to create a branched deletion intermediate. The fragment ends provide evidence for a previously uncharacterized third step: the branched DNA strand is cleaved at one of several defined sites located within 15-16 nucleotides of the IES boundary, liberating the deleted DNA in a linear form.

Fig. 1. Programmed genomic deletions in T.thermophila. (A) DNA deletions in the M and R regions. Deletions are directed by cis-acting signals located 30–50 bp outside boundaries of the deleted sequences (indicated by black half-arrows). In the M region, there are separate cis-acting signals located at left ends of the 0.6 or 0.9 kbp IES deletions. (B) A model for programmed genomic deletions. (1) The reaction is initiated by a single double-stranded cleavage at one boundary of the IES, generating DNA ends with a 4 nt 5′ extension. There is generally an adenosine residue at the 3′ chromosomal terminus. (2) The 3′ hydroxyl of the adenosine residue serves as the nucleophile in a strand transfer step at a phosphodiester bond at the opposite boundary. This step creates the macronuclear junction on one DNA strand. (3) The other strand must then be cleaved.

Fig. 2. Purification and detection of linear forms of IES elements excised from the M and R regions. (A) Technique to purify a linear IES. After electrophoresis, DNA is isolated from gel slices corresponding to fragments of different sizes. The 100 bp ladder of markers was required to follow the migration of linear forms of IESs, which are invisible because of their low abundance. (B) Linear forms of IES elements excised from the M region were detected in gel fractions corresponding to DNAs of 600 and 900 ± 50 bp in length, Linear IES elements from the R region were detected only in fractions containing DNA of ∼1.1 kbp.

Fig. 3. DNA end structure at the M3 end of the isolated 0.6 kbp linear M IES elements. (A) The structure of DNA cleavage events detected for the 0.6 kbp M region IES in an earlier study (Saveliev and Cox, 1994). A double-stranded cleavage is detected at the M3 site, while the M3′ site features a cleavage detectable in only the upper strand. (B) Characterization of the 3′ and 5′ termini at the M3 end of the isolated 0.6 kbp linear IES elements. For the 3′ termini, two separate trials are shown. For the 5′ termini, seven separate trials are shown. In total, two 3′ and five 5′ termini were detected. The origin of a third band generated during study of 3′ termini (seen just above M3′ product) is unclear. It does not generate any PCR product during re-amplification for sequencing purposes. Most likely, it is a non-specific PCR product. (C) Map of all termini detected in this and previous work. All the termini shown in (B) were sequenced to pinpoint their exact positions. The 3′ and 5′ termini at the far right are generated during an initiating double-stranded cleavage. All other termini appear to represent single-strand breaks. The known chromosomal junctions show that macronucleus-retained flanking sequences join at positions (called macronuclear or chromosomal junctions) corresponding to the 3′ termini, but not the 5′ termini.

Fig. 4. DNA ends at the M3 end of the 0.9 kbp linear M IES. (A) The structure of DNA cleavage events detected for the 0.9 kbp M region IES in an earlier study (Saveliev and Cox, 1994). Double-stranded cleavage at the left boundary of the 0.9 kbp M region can occur at either of two sites shifted by 4 bp relative to each other. Joining of macronucleus-retained DNA ends at these sites generates the three most common chromosomal junctions (Saveliev and Cox, 1994). (B) Characterization of the 3′ and 5′ termini at the M3 end of the isolated 0.9 kbp linear IES element. In total, three 3′ and five 5′ termini were detected. Each lane of each gel corresponds to an individual trial. (C) Map of the DNA termini and macronuclear junctions. All the termini shown in (B) were sequenced to pinpoint their exact positions. As in the 0.6 kbp M region, the distal 3′ and 5′ termini at the M3 end of the 0.9 kbp M region are generated during initiating double-stranded cleavage, whereas all other termini appear to be single-stranded breaks. Two new chromosomal junctions generated during the 0.9 kbp deletion in the M region are also shown. Junctions with the ‘upper’ designation were generated by an initiating DNA cleavage on the left side of the element, followed by strand transfer on the upper strand as drawn. Those with the ‘bottom’ designation underwent an initiating cleavage on the right side of the element, with strand transfer involving the bottom strand. The absence of an M1(1)M3upper junction has been tentatively attributed to the unique presence of a 3′ terminal G residue at M1, which may not function in strand transfer (Saveliev and Cox, 1996). As for the termini observed in Figure 3, each of the detected 3′ termini corresponds to defined macronuclear junctions, whereas some of the detected 5′ termini do not.

Fig. 5. Characterization of DNA ends at the M1 end of the 0.9 kbp linear M IES. (A) As determined by DNA sequencing, the slightly smeared single band represents two 3′ termini with ends only 4 bp apart. Four 5′ termini were detected and easily resolved in the gel. (B) Map of the DNA ends. All the termini were sequenced and placed on the map. Two new 5′ termini detected in this study (–10 and –16) appear to represent single-strand breaks. Each gel lane represents an individual trial.

Fig. 6. Initiating cleavage events at M3 in chromosomal DNA. (A) Protocol illustration. Chromosomal DNA was purified in 1.5% agarose gel to remove linear IES elements. The 3′ termini generated by an initiating cleavage at the M3 end were detected by TdT-mediated PCR. Only one 3′ terminus was detected, and this only in the fraction containing the chromosomal DNA fragments (DNA fragment sizes in kbp are indicated at the top of each gel lane). (B) Map of the initiating cleavage with other 3′ ends also shown. The PCR product shown in (A) was sequenced multiple times and shown to coincide with the known double-stranded cleavage at the M3 boundary. The positions of detected single-strand breaks (–3, –8, –13) are indicated to stress that an initiating cleavage occurs only at M3.

Fig. 7. A model for the late stages of programmed deletions in the M and R regions of T.thermophila. Circled step numbers correspond to those in Figure 1. The strand transfer step shown in Figure 1B creates the macronuclear junction on one strand, and also creates a new 3′ terminus on the displaced strand of the IES element. The IES element is still joined as a branch of the other strand. The branched DNA strand is then cleaved at any of several defined sites located within 15–16 nt of the boundary. This cleavage (step 3) would remove the IES element, leaving short single-strand branches (which can not anneal with the sequences on the chromosomal junction strand) in the chromosomal DNA. Subsequently, these branches would have to be removed by nucleases (a step we have no evidence for), and the resulting gap filled in by DNA polymerase. As a result of the various cleavage events, one end of the excised IES has a 4 nt 5′ extension, whereas the opposite end has a 3′ extension of up to 12 nt. The excised IES may be quickly packaged into subnuclear structures together with thousands of other deleted DNA fragments to avoid their re-integration into chromosomal DNA. These could be transported outside of the developing macronucleus and digested to nucleotides.

Fig. 8. Map of the oligonucleotides used in this work. Sequences are listed in ITable I. Arrows indicate 3′ ends. Maps are not drawn to scale, and are intended only to indicate the relative linear order of the sequences to which the oligos pair. Some oligos used in specialized procedures at DNA ends, and which do not pair with any sequence in the IES elements [A, A(C) and J], are not shown.