The incorporation and creation of modified nucleobases in DNA have profound effects on genome function. We describe methods for mapping positions and local content of modified DNA nucleobases in genomic DNA. We combined in vitro nucleobase excision with massively parallel DNA sequencing (Excision-seq) to determine the locations of modified nucleobases in genomic DNA. We applied the Excision-seq method to map uracil in E. coli and budding yeast and discovered significant variation in uracil content, wherein uracil is excluded from the earliest and latest replicating regions of the genome, possibly driven by changes in nucleotide pool composition. We also used Excision-seq to identify sites of pyrimidine dimer formation induced by UV light exposure, where the method could distinguish between sites of cyclobutane and 6-4 photoproduct formation. These UV mapping data enabled analysis of local sequence bias around pyrimidine dimers and suggested a preference for an adenosine downstream from 6-4 photoproducts. The Excision-seq method is broadly applicable for high precision, genome-wide mapping of modified nucleobases with cognate repair enzymes.[Supplemental material is available for this article.]Many different modifications of the four primary DNA nucleobases expand the chemical diversity of DNA and have profound effects on genome function. Intrinsic modifications (e.g., 5-methylcytosine and uracil) are integral to genetic and epigenetic regulation. Extrinsic modifications (e.g., pyrimidine dimers and nucleobase oxidation) arise from environmental exposures and can initiate aberrant cell growth or death. A detailed understanding of intrinsic and extrinsic nucleobase modification is necessary for a complete view of genetic and epigenetic regulation, but a global picture of how nucleobase modifications are created, maintained, and repaired, and how their spatial distribution impacts genome function, is lacking.Incorporation of uracil into DNA creates detrimental or beneficial mutations, depending on context. To sustain DNA replication, cells must synthesize or scavenge precursors to accumulate a pool of nucleotide triphosphates. A key step of thymidine triphosphate (TTP) synthesis is catalyzed by thymidylate synthase, which converts dUMP to dTMP using tetrahydrofolate as a methyl donor. One branch of the TTP biosynthetic pathway uses dUTP as an intermediate, which can be incorporated into DNA in the form of A:U base pairs. The upstream production of dUMP is catalyzed by deamination of dCMP by deoxycytidylate deaminase or pyrophosphorolysis of dUTP by the dUTP pyrophosphatase (Dut1). Dut1 is essential for viability and normal nucleotide metabolism: In the absence of Dut1, cells simultaneously accumulate dUTP and deplete TTP pools (Gadsden et al. 1993), causing a futile cycle of uracil incorporation and repair that leads to extensive DNA damage (Kavli et al. 2007).Uracil in DNA is removed by uracil DNA glycosylase (UDG) enzymes, which scan double-stranded DNA for uracil and cleave its glycosidic bond (Krokan et al. 200...

Turnover of the branched RNA intermediates and products of pre-mRNA splicing is mediated by the lariat-debranching enzyme Dbr1. We characterized a homolog of Dbr1 from Saccharomyces cerevisiae, Drn1/Ygr093w, that has a pseudometallophosphodiesterase domain with primary sequence homology to Dbr1 but lacks essential active site residues found in Dbr1. Whereas loss of Dbr1 results in lariat-introns failing broadly to turnover, loss of Drn1 causes low levels of lariat-intron accumulation. Conserved residues in the Drn1 C-terminal CwfJ domains, which are not present in Dbr1, are required for efficient intron turnover. Drn1 interacts with Dbr1, components of the Nineteen Complex, U2 snRNA, branched intermediates, and products of splicing. Drn1 enhances debranching catalyzed by Dbr1 in vitro, but does so without significantly improving the affinity of Dbr1 for branched RNA. Splicing carried out in in vitro extracts in the absence of Drn1 results in an accumulation of branched splicing intermediates and products released from the spliceosome, likely due to less active debranching, as well as the promiscuous release of cleaved 5 ′ -exon. Drn1 enhances Dbr1-mediated turnover of lariat-intermediates and lariat-intron products, indicating that branched RNA turnover is regulated at multiple steps during splicing.

RNA cleavage by some endoribonucleases and self-cleaving ribozymes produces RNA fragments with 5′-hydroxyl (5′-OH) and 2′,3′-cyclic phosphate termini. To identify 5′-OH RNA fragments produced by these cleavage events, we exploited the unique ligation mechanism of Escherichia coli RtcB RNA ligase to attach an oligonucleotide linker to RNAs with 5′-OH termini, followed by steps for library construction and analysis by massively parallel DNA sequencing. We applied the method to RNA from budding yeast and captured known 5′-OH fragments produced by tRNA Splicing Endonuclease (SEN) during processing of intron-containing pre-tRNAs and by Ire1 cleavage of HAC1 mRNA following induction of the unfolded protein response (UPR). We identified numerous novel 5′-OH fragments derived from mRNAs: some 5′-OH mRNA fragments were derived from single, localized cleavages, while others were likely produced by multiple, distributed cleavages. Many 5′-OH fragments derived from mRNAs were produced upstream of codons for highly electrostatic peptides, suggesting that the fragments may be generated by co-translational mRNA decay. Several 5′-OH RNA fragments accumulated during the induction of the UPR, some of which share a common sequence motif that may direct cleavage of these mRNAs. This method enables specific capture of 5′-OH termini and complements existing methods for identifying RNAs with 2′,3′-cyclic phosphate termini.

RNA repair enzymes catalyze rejoining of an RNA molecule after cleavage of phosphodiester linkages. RNA repair in budding yeast is catalyzed by two separate enzymes that process tRNA exons during their splicing and mRNA exons during activation of the unfolded protein response (UPR). The RNA ligase Trl1 joins 2',3'-cyclic phosphate and 5'-hydroxyl RNA fragments, creating a phosphodiester linkage with a 2'-phosphate at the junction. The 2'-phosphate is removed by the 2'-phosphotransferase Tpt1. We bypassed the essential functions of and in budding yeast by expressing "prespliced," intronless versions of the 10 normally intron-containing tRNAs, indicating this repair pathway does not have additional essential functions. Consistent with previous studies, expression of intronless tRNAs failed to rescue the growth of cells with deletions in components of the SEN complex, implying an additional essential role for the splicing endonuclease. TheΔ and Δ mutants accumulate tRNA and splicing intermediates indicative of RNA repair defects and are hypersensitive to drugs that inhibit translation. Failure to induce the unfolded protein response in Δ cells grown with tunicamycin is lethal owing to their inability to ligate after its cleavage by Ire1. In contrast, Δ mutants grow in the presence of tunicamycin despite reduced accumulation of spliced mRNA. We optimized a PCR-based method to detect RNA 2'-phosphate modifications and show they are present on ligated mRNA. These RNA repair mutants enable new studies of the role of RNA repair in cellular physiology.

The EphA4 receptor tyrosine kinase is involved in numerous cell-signalling activities during embryonic development. EphA4 has the ability to bind to both types of ephrin ligands, the ephrinAs and ephrinBs. The C57BL/6J-Epha4rb-2J/GrsrJ strain, denoted Epha4(rb-2J/rb-2J), is a spontaneous mouse mutant that arose at The Jackson Laboratory. These mutants exhibited a synchronous hind limb locomotion defect or "hopping gait" phenotype, which is also characteristic of EphA4 null mice. Genetic complementation experiments suggested that Epha4(rb-2J) corresponds to an allele of EphA4, but details of the genomic defect in this mouse mutant are currently unavailable. We found a single base-pair deletion in exon 9 resulting in a frame shift mutation that subsequently resulted in a premature stop codon. Analysis of the predicted structure of the truncated protein suggests that both the kinase and sterile α motif (SAM) domains are absent. Definitive determination of genotype is needed for experimental studies of mice carrying the Epha4(rb-2J) allele, and we have also developed a method to ease detection of the mutation through RFLP. Eph-ephrin family members are reportedly expressed as numerous isoforms. Hence, delineation of the specific mutation in EphA4 in this strain is important for further functional studies, such as protein-protein interactions, immunostaining and gene compensatory studies, investigating the mechanism underlying the effects of altered function of Eph family of receptor tyrosine kinases on phenotype.

We have developed a cost-effective, highly parallel method for purification and functionalization of 5′-labeled oligonucleotides. The approach is based on 5′-hexa-His phase tag purification, followed by exchange of the hexa-His tag for a functional group using reversible reaction chemistry. These methods are suitable for large-scale (micromole to millimole) production of oligonucleotides and are amenable to highly parallel processing of many oligonucleotides individually or in high complexity pools. Examples of the preparation of 5′-biotin, 95-mer, oligonucleotide pools of >40K complexity at micromole scale are shown. These pools are prepared in up to ~16% yield and 90–99% purity. Approaches for using this method in other applications are also discussed.