Mathy, N. | bioRxiv Science

bioRxiv2024

A development-specific POLX essential for programmed genome rearrangement in Paramecium tetraurelia

During the sexual cycle, programmed genome rearrangement (PGR) in Paramecium tetraurelia involves the non-homologous end joining (NHEJ) DNA repair pathway to eliminate specific germinal Internal Eliminated Sequences (IESs) from the newly developing somatic nucleus. In addition to the core NHEJ factors Ku70/80 and Xrcc4/Lig4, additional enzymes are required to process the 4-base 5-protruding ends generated following DNA cleavage at IES boundaries, prior to their ligation. Here, we report that PolX (a,b,c,d), four P. tetraurelia distant orthologs of the human Pol{lambda} DNA polymerase, are involved in repair of IES excision junctions. During rearrangements, PolX-depleted cells accumulate genome-wide errors, such as unrepaired double-strand breaks, 1-nucleotide deletions and IES retention. Although all PolX paralogs can process DNA ends, two of them (PolXa&b) are induced during PGR and have acquired tight nuclear anchoring properties through their N-terminal region, which contains a predicted BRCT domain. Finally, we show that PolXa accumulates in nuclear foci together with other NHEJ proteins and the Dicer-like enzyme Dcl5, which is involved in the biogenesis of IES-specific small RNAs. We propose that these "DNA repair foci" correspond to the sites where IES concatemers, a by-product of IES excision, are ligated together to produce the precursors of iesRNAs.

molecular biology↗

bioRxiv2022

Developmental timing of programmed DNA elimination in Paramecium tetraurelia recapitulates germline transposon evolutionary dynamics

With its nuclear dualism, the ciliate Paramecium constitutes an original model to study how host genomes cope with transposable elements (TEs). P. tetraurelia harbors two germline micronuclei (MIC) and a polyploid somatic macronucleus (MAC) that develops from the MIC at each sexual cycle. Throughout evolution, the MIC genome has been continuously colonized by TEs and related sequences that are removed from the somatic genome during MAC development. Whereas TE elimination is generally imprecise, excision of [~]45,000 TE-derived Internal Eliminated Sequences (IESs) is precise, allowing for functional gene assembly. Programmed DNA elimination is concomitant with genome amplification. It is guided by non-coding RNAs and repressive chromatin marks. A subset of IESs is excised independently of this epigenetic control, raising the question of how IESs are targeted for elimination. To gain insight into the determinants of IES excision, we established the developmental timing of DNA elimination genome-wide by combining fluorescence-assisted nuclear sorting with next-generation sequencing. Essentially all IESs are excised within only one endoreplication round (32C to 64C), while TEs are eliminated at a later stage. We show that time, rather than replication, controls the progression of DNA elimination. We defined four IES classes according to excision timing. The earliest excised IESs tend to be independent of epigenetic factors, display strong sequence signals at their ends and originate from the most ancient integration events. We conclude that old IESs have been optimized during evolution for early and accurate excision, by acquiring stronger sequence determinants and escaping epigenetic control.

genomics↗