Bryan D Suzi, Ransom Monica, Adane Biniam, York Kerri, Hesselberth Jay R
Department of Biochemistry and Molecular Genetics, Program in Molecular Biology, University of Colorado School of Medicine, Aurora, Colorado 80045, USA.
Department of Biochemistry and Molecular Genetics, Program in Molecular Biology, University of Colorado School of Medicine, Aurora, Colorado 80045, USA
Genome Res. 2014 Sep;24(9):1534-42. doi: 10.1101/gr.174052.114. Epub 2014 Jul 11.
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.
DNA中修饰核苷酸碱基的掺入和产生对基因组功能有深远影响。我们描述了在基因组DNA中定位修饰的DNA核苷酸碱基位置和局部含量的方法。我们将体外核苷酸碱基切除与大规模平行DNA测序（切除测序）相结合，以确定基因组DNA中修饰核苷酸碱基的位置。我们应用切除测序方法绘制大肠杆菌和芽殖酵母中的尿嘧啶图谱，并发现尿嘧啶含量存在显著差异，其中尿嘧啶被排除在基因组最早和最晚复制区域之外，这可能是由核苷酸库组成的变化驱动的。我们还使用切除测序来鉴定紫外线照射诱导的嘧啶二聚体形成位点，该方法可以区分环丁烷和6-4光产物形成位点。这些紫外线图谱数据能够分析嘧啶二聚体周围的局部序列偏差，并表明在6-4光产物下游对腺苷有偏好。切除测序方法广泛适用于使用同源修复酶对修饰核苷酸碱基进行高精度全基因组图谱绘制。
