High resolution mapping of modified DNA nucleobases using excision repair enzymes

Abstract

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...

Genome-wide analysis of human global and transcription-coupled excision repair of UV damage at single-nucleotide resolution

“…For TC at position 19-20 in the (6-4)PP XR-seq fragments, there is a pronounced preference for C 5 ′ and A 3 ′ to the putative photoproduct site (P < 0.02) (Materials and Methods; Supplemental Table 4). These preferences are consistent with previous reports on sequence effects on photoproduct formation (Mitchell et al 1992;Bryan et al 2014).…”

“…Thus, any method used to map DNA repair must yield a high signal to noise ratio and high sensitivity. Although genome-wide maps of UV damage distribution (Teng et al 2011;Bryan et al 2014;Zavala et al 2014;Powell et al 2015) and single-gene analyses of excision repair in human cells and yeast are available (Pfeifer et al 1991;Tornaletti and Pfeifer 1994;Denissenko et al 1996;Li et al 2000Li et al , 2014, methods for genome-wide detection of excision repair at nucleotide resolution have not been reported.…”

Chromosomal landscape of UV damage formation and repair at single-nucleotide resolution

“…S8). Such a nucleosome photo-footprint was identified in mammalian chromatin nearly 30 y ago (11), but had been missed by previous genome-wide studies of CPD-formation (15,16,18,25). Nucleosomal DNA with an inward rotational setting is likely protected from UV damage as a result of constraints on DNA bending and flexibility imposed by the nucleosome structure (22).…”

“…However, the low resolution of these studies precluded analysis of how chromatin or transcription factors affect CPD formation or repair throughout the genome. More recently, a high-throughput sequencing method called Excision-seq was used to map CPDs and 6-4PPs in the yeast genome at single-nucleotide resolution (18). This method did confirm the expected DNA sequence preferences for CPD and 6-4PP formation (18), but there was no reported effect of chromatin or DNA-binding proteins on UV damage formation.…”

“…More recently, a high-throughput sequencing method called Excision-seq was used to map CPDs and 6-4PPs in the yeast genome at single-nucleotide resolution (18). This method did confirm the expected DNA sequence preferences for CPD and 6-4PP formation (18), but there was no reported effect of chromatin or DNA-binding proteins on UV damage formation. Moreover, Excision-seq requires Significance UV-induced DNA lesions are an important contributor to melanomas and other skin cancers.…”