Highly Precise and Developmentally Programmed Genome Assembly in Paramecium Requires Ligase IV–Dependent End Joining

Abstract

During the sexual cycle of the ciliate Paramecium, assembly of the somatic genome includes the precise excision of tens of thousands of short, non-coding germline sequences (Internal Eliminated Sequences or IESs), each one flanked by two TA dinucleotides. It has been reported previously that these genome rearrangements are initiated by the introduction of developmentally programmed DNA double-strand breaks (DSBs), which depend on the domesticated transposase PiggyMac. These DSBs all exhibit a characteristic geometry, with 4-base 5′ overhangs centered on the conserved TA, and may readily align and undergo ligation with minimal processing. However, the molecular steps and actors involved in the final and precise assembly of somatic genes have remained unknown. We demonstrate here that Ligase IV and Xrcc4p, core components of the non-homologous end-joining pathway (NHEJ), are required both for the repair of IES excision sites and for the circularization of excised IESs. The transcription of LIG4 and XRCC4 is induced early during the sexual cycle and a Lig4p-GFP fusion protein accumulates in the developing somatic nucleus by the time IES excision takes place. RNAi–mediated silencing of either gene results in the persistence of free broken DNA ends, apparently protected against extensive resection. At the nucleotide level, controlled removal of the 5′-terminal nucleotide occurs normally in LIG4-silenced cells, while nucleotide addition to the 3′ ends of the breaks is blocked, together with the final joining step, indicative of a coupling between NHEJ polymerase and ligase activities. Taken together, our data indicate that IES excision is a “cut-and-close” mechanism, which involves the introduction of initiating double-strand cleavages at both ends of each IES, followed by DSB repair via highly precise end joining. This work broadens our current view on how the cellular NHEJ pathway has cooperated with domesticated transposases for the emergence of new mechanisms involved in genome dynamics.

Sources of DNA Double-Strand Breaks and Models of Recombinational DNA Repair

“…In particular, thousands of short noncoding germline sequences, called internal eliminated sequences, are spliced out (Betermier 2004). These developmentally programmed DNA DSBs depend on the domesticated transposase PiggyMac (Baudry et al 2009) and are repaired using components of the nonhomologous end-joining (NHEJ) pathway (Kapusta et al 2011).…”

SUMOylation Is Developmentally Regulated and Required for Cell Pairing during Conjugation in Tetrahymena thermophila

“…The NHEJ pathway is believed to be the key mechanism for repair of double-stranded DNA (dsDNA) breaks generated during programmed DNA rearrangements that occur naturally during conjugation in ciliates (15). In Paramecium tetraurelia, the RNAi-induced silencing of ligase IV (a partner of XRCC4 in NHEJ) during conjugation results in the persistence of free broken ends during genome reorganization (53). The increasing links between NHEJ pathway proteins and genome reorganization during ciliate conjugation suggest potential targets for SUMOylation.…”

Lack of Association Between LIG4 Gene Polymorphisms and the Risk of Breast Cancer: A HuGE Review and Meta-analysis

“…The protein encoded by this gene is an ATP-dependent DNA ligase that joins DSB during the NHEJ repair pathway (Kapusta et al, 2011). LIG4 forms a complex with XRCC4, and further interacts with the DNA-dependent protein kinase (DNA-PK) and XLF, which are also required for NHEJ (Symington and Gautier, 2011).…”