Repair of UV-induced DNA lesions in natural Saccharomyces cerevisiae telomeres is moderated by Sir2 and Sir3, and inhibited by yKu-Sir4 interaction.

Ultraviolet light (UV) causes DNA damage that is removed by nucleotide excision repair (NER). UV-induced DNA lesions must be recognized and repaired in nucleosomal DNA, higher order structures of chromatin and within different nuclear sub-compartments. Telomeric DNA is made of short tandem repeats located at the ends of chromosomes and their maintenance is critical to prevent genome instability. In Saccharomyces cerevisiae the chromatin structure of natural telomeres is distinctive and contingent to telomeric DNA sequences. Namely, nucleosomes and Sir proteins form the heterochromatin like structure of X-type telomeres, whereas a more open conformation is present at Y'-type telomeres. It is proposed that there are no nucleosomes on the most distal telomeric repeat DNA, which is bound by a complex of proteins and folded into higher order structure. How these structures affect NER is poorly understood. Our data indicate that the X-type, but not the Y'-type, sub-telomeric chromatin modulates NER, a consequence of Sir protein-dependent nucleosome stability. The telomere terminal complex also prevents NER, however, this effect is largely dependent on the yKu-Sir4 interaction, but Sir2 and Sir3 independent.

Immuno-capture of UVDE generated 3'-OH ends at UV photoproducts.

A strategy amenable to the genome-wide study of DNA damage and repair kinetics is described. The ultraviolet damage endonuclease (UVDE) generates 3'-OH ends at the two major UV induced DNA lesions, cyclobutane pyrimidine dimers (CPDs) and 6,4 pyrimidine-pyrimidone dimers (6,4 PPs), allowing for their capture after biotin end-labeling. qPCR amplification of biotinylated DNA enables parallel measuring of DNA damage in several loci, which can then be combined with high-throughput screening of cell survival to test genotoxic reagents. Alternatively, a library of captured sequences could be generated for a genome wide study of damage sites and large-scale assessment of repair kinetics in different regions of the genome, using next-generation sequencing. The assay is suitable to study any DNA lesion that can be converted into 3'-OH by UVDE, or other enzymes. Toward these goals, we compared UVDE with the classical T4 endonuclease V (T4V) assay. We showed that there is a linear correlation between UV dose, 3'-OH formation and capture by immunoprecipitation, together with its potential application for in vivo studies.

The nuclear poly(A)-binding protein interacts with the exosome to promote synthesis of noncoding small nucleolar RNAs.

Poly(A)-binding proteins (PABPs) are important to eukaryotic gene expression. In the nucleus, the PABP PABPN1 is thought to function in polyadenylation of pre-mRNAs. Deletion of fission yeast pab2, the homolog of mammalian PABPN1, results in transcripts with markedly longer poly(A) tails, but the nature of the hyperadenylated transcripts and the mechanism that leads to RNA hyperadenylation remain unclear. Here we report that Pab2 functions in the synthesis of noncoding RNAs, contrary to the notion that PABPs function exclusively on protein-coding mRNAs. Accordingly, the absence of Pab2 leads to the accumulation of polyadenylated small nucleolar RNAs (snoRNAs). Our findings suggest that Pab2 promotes poly(A) tail trimming from pre-snoRNAs by recruiting the nuclear exosome. This work unveils a function for the nuclear PABP in snoRNA synthesis and provides insights into exosome recruitment to polyadenylated RNAs.

A methyltransferase-independent function for Rmt3 in ribosomal subunit homeostasis.

Schizosaccharomyces pombe Rmt3 is a member of the protein-arginine methyltransferase (PRMT) family and is the homolog of human PRMT3. We previously characterized Rmt3 as a ribosomal protein methyltransferase based on the identification of the 40 S Rps2 (ribosomal protein S2) as a substrate of Rmt3. RMT3-null cells produce nonmethylated Rps2 and show mis-regulation of the 40 S/60 S ribosomal subunit ratio due to a small subunit deficit. For this study, we have generated a series of RMT3 alleles that express various amino acid substitutions to characterize the functional domains of Rmt3 in Rps2 binding, Rps2 arginine methylation, and small ribosomal subunit production. Notably, catalytically inactive versions of Rmt3 restored the ribosomal subunit imbalance detected in RMT3-null cells. Consistent with a methyltransferase-independent function for Rmt3 in small ribosomal subunit production, the expression of an Rps2 variant in which the identified methylarginine residues were substituted with lysines showed normal levels of 40 S subunit. Importantly, substitutions within the zinc finger domain of Rmt3 that abolished Rps2 binding did not rescue the 40 S ribosomal subunit deficit of RMT3-null cells. Our findings suggest that the Rmt3-Rps2 interaction, rather than Rps2 methylation, is important for the function of Rmt3 in the regulation of small ribosomal subunit production.

Nucleotide excision repair and photolyase repair of UV photoproducts in nucleosomes: assessing the existence of nucleosome and non-nucleosome rDNA chromatin in vivo.

The genome is organized into nuclear domains, which create microenvironments that favor distinct chromatin structures and functions (e.g., highly repetitive sequences, centromeres, telomeres, noncoding sequences, inactive genes, RNA polymerase II and III transcribed genes, and the nucleolus). Correlations have been drawn between gene silencing and proximity to a heterochromatic compartment. At the other end of the scale are ribosomal genes, which are transcribed at a very high rate by RNA polymerase I (~60% of total transcription), have a loose chromatin structure, and are clustered in the nucleolus. The rDNA sequences have 2 distinct structures: active rRNA genes, which have no nucleosomes; and inactive rRNA genes, which have nucleosomes. Like DNA transcription and replication, DNA repair is modulated by the structure of chromatin, and the kinetics of DNA repair vary among the nuclear domains. Although research on DNA repair in all chromosomal contexts is important to understand the mechanisms of genome maintenance, this review focuses on nucleotide excision repair and photolyase repair of UV photoproducts in the first-order packing of DNA in chromatin: the nucleosome. In addition, it summarizes the studies that have demonstrated the existence of the 2 rDNA chromatins, and the way this feature of the rDNA locus allows for direct comparison of DNA repair in 2 very different structures: nucleosome and non-nucleosome DNA.