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...

DDK, a conserved serine-threonine protein kinase composed of a regulatory subunit, Dbf4, and a catalytic subunit, Cdc7, is essential for DNA replication initiation during S phase of the cell cycle through MCM2-7 helicase phosphorylation. The biological significance of DDK is well characterized, but the full mechanism of how DDK associates with substrates remains unclear. Cdc7 is bound to chromatin in the Saccharomyces cerevisiae genome throughout the cell cycle, but there is little empirical evidence as to specific Cdc7 binding locations. Using biochemical and genetic techniques, this study investigated the specific localization of Cdc7 on chromatin. The Calling Cards method, using Ty5 retrotransposons as a marker for DNA-protein binding, suggests Cdc7 kinase is preferentially bound to genomic DNA known to replicate early in S phase, including centromeres and origins of replication. We also discovered Cdc7 binding throughout the genome, which may be necessary to initiate other cellular processes, including meiotic recombination and translesion synthesis. A kinase dead Cdc7 point mutation increases the Ty5 retrotransposon integration efficiency and a 55-amino acid C-terminal truncation of Cdc7, unable to bind Dbf4, reduces Cdc7 binding suggesting a requirement for Dbf4 to stabilize Cdc7 on chromatin during S phase. Chromatin immunoprecipitation demonstrates that Cdc7 binding near specific origins changes during S phase. Our results suggest a model where Cdc7 is loosely bound to chromatin during G 1 . At the G 1 /S transition, Cdc7 binding to chromatin is increased and stabilized, preferentially at sites that may become origins, in order to carry out a variety of cellular processes. KEYWORDSreplication kinase calling cards chromatin origins DNA replication in eukaryotes is a tightly regulated process that ensures the genome is duplicated once and only once during the cell cycle. Failure to control replication mechanisms leads to chromosome instability, aneuploidy, and mutations, all hallmarks of many human diseases (Rossbach and Sclafani 2016). DNA replication is initiated at specific segments of genomic DNA, termed origins of replication, during S phase of the cell cycle. In budding yeast, Saccharomyces cerevisiae, origins are known as ARSs (autonomous replicating sequences) and contain an 11 bp consensus sequence termed the ACS (ARS consensus sequence) that serves as a docking site for replisome proteins (Sclafani and Holzen 2007). Origins are classified by two key characteristics: efficiency and timing. Origin efficiency is determined by how frequently the origin initiates replication and whether it is used in a population of cells during every cellular division. Origin timing is a temporal reflection of when a given origin initiates replication during S phase. Origins can initiate replication throughout S phase and the time they initiate is quantified by the T rep (timing of replication), the time it takes to duplicate 50% of the DNA (Raghuraman et al. 2001;Yabuki et al. 2002).Initiation of DNA replic...

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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.