Functional diversification of Paramecium Ku80 paralogs safeguards genome integrity during precise programmed DNA elimination

Abstract

Gene duplication and diversification drive the emergence of novel functions during evolution. Because of whole genome duplications, ciliates from the Paramecium aurelia group constitute a remarkable system to study the evolutionary fate of duplicated genes. Paramecium species harbor two types of nuclei: a germline micronucleus (MIC) and a somatic macronucleus (MAC) that forms from the MIC at each sexual cycle. During MAC development, 45,000 germline Internal Eliminated Sequences (IES) are excised precisely from the genome through a 'cut-and-close' mechanism. Here, we have studied the P. tetraurelia paralogs of KU80, which encode a key DNA double-strand break repair factor involved in non-homologous end joining. The three KU80 genes have different transcription patterns, KU80a and KU80b being constitutively expressed, while KU80c is specifically induced during MAC development. Immunofluorescence microscopy and high-throughput DNA sequencing revealed that Ku80c stably anchors the PiggyMac (Pgm) endonuclease in the developing MAC and is essential for IES excision genome-wide, providing a molecular explanation for the previously reported Ku-dependent licensing of DNA cleavage at IES ends. Expressing Ku80a under KU80c transcription signals failed to complement a depletion of endogenous Ku80c, indicating that the two paralogous proteins have distinct properties. Domain-swap experiments identified the α/β domain of Ku80c as the major determinant for its specialized function, while its C-terminal part is required for excision of only a small subset of IESs located in IES-dense regions. We conclude that Ku80c has acquired the ability to license Pgm-dependent DNA cleavage, securing precise DNA elimination during programmed rearrangements. The present study thus provides novel evidence for functional diversification of genes issued from a whole-genome duplication.

The Paramecium histone chaperone Spt16-1 is required for Pgm endonuclease function in programmed genome rearrangements

“…To quantify the effects of SPT16-1 KD on IES retention, we used sequencing data from non-silenced autogamous cells of the same strain, as a control [24]. Excision of all IESs is altered and 98.6% (44,334) IESs are statistically significantly retained in the developing MAC after SPT16-1 KD, similar to the 99% (44,491) observed after PGM KD [36] (S7 Fig). We measured IES retention at each IES boundary, using the boundary score method [36].…”

“…tetraurelia , a specialized Ku70/Ku80 heterodimer acts upstream of DNA cleavage, instead of downstream. Indeed, the depletion of the developmental-specific Ku80 paralog Ku80c abolished DNA cleavage at IES ends, resulting in retention of all IESs in the new MAC [ 16 , 44 ], as for Spt16-1 and Pgm depletion. Furthermore, similar to Spt16-1 being essential for Pgm nuclear localization, Ku80c is required for anchoring the Pgm endonuclease in the developing MAC [ 44 ].…”

Massive colonization of protein-coding exons by selfish genetic elements in Paramecium germline genomes

“…IESs are invariably bounded by two 5 0 -TA-3 0 dinucleotides, one of which is left at the junction in the MAC genome after excision. IES excision in the developing MAC is initiated by DNA double-strand breaks at IES ends by the endonuclease PiggyMac (Pgm) assisted by other proteins, which are likely part of the excision machinery or interact with it [6][7][8][9]. Despite significant progress in characterization of the mechanisms underlying IES elimination, the evolutionary origin of IESs remains mysterious.…”

The Multifaceted Roles of Ku70/80

“…The Ku70a/80c heterodimer co-purifies with PiggyMac and is essential for the licensing of its endonuclease [ 169 ]. Recently, immunofluorescence microscopy and high-throughput DNA sequencing revealed that Ku80c stably anchors the PiggyMac (Pgm) endonuclease through an interaction on the α/β domain of Ku80c [ 170 ]. The ciliate model suggests a tight coupling between DSB introduction and repair with a central role of Ku [ 171 ].…”