Evolutionary origins of apoB mRNA editing: Catalysis by a cytidine deaminase that has acquired a novel RNA-binding motif at its active site

AbstractThe site-specific C to U editing of apolipoprotein B100 (apoB100) mRNA requires a 27 kDa protein (p27) with homology to cytidine deaminase. Here, we show that p27 is a zinc-containing deaminase, which operates catalytically like the E. coli enzyme that acts on monomeric substrate. In contrast with the bacterial enzyme that does not bind RNA, p27 interacts with its polymeric apoB m RNA substrate at AU sequences adjacent to the editing site. This interaction is necessary for editing. RNA binding is mediated through amino acid residues involved in zinc coordination, in proton shuttling, and in forming the αβα structure that encompasses the active site. However, certain mutations that inactivate the enzyme do not affect RNA binding. Thus, RNA binding does not require a catalytically active site. The acquisition of polymeric substrate binding provides a route for the evolution of this editing enzyme from one that acts on monomeric substrates

Rtt107 Phosphorylation Promotes Localisation to DNA Double-Stranded Breaks (DSBs) and Recombinational Repair between Sister Chromatids

Efficient repair of DNA double-stranded breaks (DSB) requires a coordinated response at the site of lesion. Nucleolytic resection commits repair towards homologous recombination, which preferentially occurs between sister chromatids. DSB resection promotes recruitment of the Mec1 checkpoint kinase to the break. Rtt107 is a target of Mec1 and serves as a scaffold during repair. Rtt107 plays an important role during rescue of damaged replication forks, however whether Rtt107 contributes to the repair of DSBs is unknown. Here we show that Rtt107 is recruited to DSBs induced by the HO endonuclease. Rtt107 phosphorylation by Mec1 and its interaction with the Smc5–Smc6 complex are both required for Rtt107 loading to breaks, while Rtt107 regulators Slx4 and Rtt101 are not. We demonstrate that Rtt107 has an effect on the efficiency of sister chromatid recombination (SCR) and propose that its recruitment to DSBs, together with the Smc5–Smc6 complex is important for repair through the SCR pathway

Cdc14 phosphatase promotes segregation of telomeres through repression of RNA polymerase II transcription

Kinases and phosphatases regulate messenger RNA synthesis through post-translational modification of the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (ref. 1). In yeast, the phosphatase Cdc14 is required for mitotic exit2,3 and for segregation of repetitive regions4. Cdc14 is also a subunit of the silencing complex RENT (refs 5, 6), but no roles in transcriptional repression have been described. Here we report that inactivation of Cdc14 causes silencing defects at the intergenic spacer sequences of ribosomal genes during interphase and at Y′ repeats in subtelomeric regions during mitosis. We show that the role of Cdc14 in silencing is independent of the RENT deacetylase subunit Sir2. Instead, Cdc14 acts directly on RNA polymerase II by targeting CTD phosphorylation at Ser 2 and Ser 5. We also find that the role of Cdc14 as a CTD phosphatase is conserved in humans. Finally, telomere segregation defects in cdc14 mutants4 correlate with the presence of subtelomeric Y′ elements and can be rescued by transcriptional inhibition of RNA polymerase II

Smc5-Smc6 mediate DNA double-strand-break repair by promoting sister-chromatid recombination

DNA double-strand breaks (DSB) can arise during DNA replication, or after exposure to DNA-damaging agents, and their correct repair is fundamental for cell survival and genomic stability. Here, we show that the Smc5-Smc6 complex is recruited to DSBs de novo to support their repair by homologous recombination between sister chromatids. In addition, we demonstrate that Smc5-Smc6 is necessary to suppress gross chromosomal rearrangements. Our findings show that the Smc5-Smc6 complex is essential for genome stability as it promotes repair of DSBs by error-free sisterchromatid recombination (SCR), thereby suppressing inappropriate non-sister recombination events