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.

Genome-wide mapping of regions preferentially targeted by the human DNA-cytosine deaminase APOBEC3A using uracil-DNA pulldown and sequencing

“…Although there is no established method for the mapping of uracils in DNA, Bryan et al (29) described two closely related methods to map uracils in DNA that depend on the generation of double-strand breaks at uracils by combining Ung with endonuclease IV, and have used it to map uracils in the HIV-1 genome (30). This method requires that the DNA is highly uracilated (Ͼ1% of cytosines converted to uracil) (29,31) and is not applicable to situations where expression of AID/APOBEC enzymes cause only small increases in genomic uracil levels (a few uracils per 10 6 bp) (32). A different method replaces the uracil with a biotinylated uracil or cytosine using complete BER and uses the biotin tag to pulldown the DNA fragments for sequencing (33) and requires a large number of biochemical steps ( Fig.…”

Detection of Genomic Uracil Patterns

“…Indeed, the same group used this method addressing the 10 kb-size HIV genomes from different in vitro infected immune cells showing uniformly distributed uracilation of the proviral genome [ 100 ]. Although this method was also extended to the mapping of other DNA base modifications [ 111 ] and cited by reviews or other research papers, Excision-seq has not yet become widely used to characterize other biological systems. In one case, pre-digestion Excision-seq was applied as a complimentary technique to support the results from dU-seq (which will be discussed later [ 112 ]).…”

Genome-wide mapping of genomic DNA damage: methods and implications

“…CPD-Seq and Excision-Seq have been employed to map UV-induced CPD in the yeast genome at a single-nucleotide resolution [ 61 , 62 ]. These two methods differ from each other in the enzymes used to recognise the CPD site.…”

“…This approach combines the use of NER enzymes to excise CPD-containing DNA fragments, from the human genome, with DDIP performed using antibodies against CPD [ 60 ]. CPD-Seq and Excision-Seq have been employed to map UV-induced CPD in the yeast genome at a single-nucleotide resolution [ 61 , 62 ]. These two methods differ from each other in the enzymes used to recognise the CPD site.…”