{"368191":{"#nid":"368191","#data":{"type":"news","title":"Ribose-seq identifies and locates ribonucleotides in genomic DNA","body":[{"value":"\u003Cp\u003ERibonucleotides, units of RNA, can become embedded in genomic DNA during processes such as DNA replication and repair, affecting the stability of the genome by contributing to DNA fragility and mutability. Scientists have known about the presence of ribonucleotides in DNA, but until now had not been able to determine exactly what they are and where they are located in the DNA sequences.\u003C\/p\u003E\u003Cp\u003ENow, researchers have developed and tested a new technique known as ribose-seq that allows them to determine the full profile of ribonucleotides embedded in genomic DNA. Using ribose-seq, they have found widespread but not random incorporation and \u201chotspots\u201d where the RNA insertions accumulate in the nuclear and mitochondrial DNA of a commonly-studied species of budding yeast. Ribose-seq could be used to locate ribonucleotides in the DNA of a wide range of other organisms, including that of humans.\u003C\/p\u003E\u003Cp\u003E\u201cRibonucleotides are the most abundant non-standard nucleotides that can be found in DNA, but until now there has not been a system to determine where they are located in the DNA, or to identify specifically which type they are,\u201d said Francesca Storici, an associate professor in the School of Biology at the Georgia Institute of Technology. \u201cBecause they change the way that DNA works, in both its structure and function, it is important to know their identity and their sites of genomic incorporation.\u201d\u003C\/p\u003E\u003Cp\u003EA description of the ribose-seq method and what it discovered in the DNA of the budding yeast species \u003Cem\u003ESaccharomyces cerevisiae\u003C\/em\u003E were reported January 26 in the journal \u003Cem\u003ENature Methods\u003C\/em\u003E. The findings resulted from collaboration between researchers in Storici\u2019s laboratory at Georgia Tech \u2013 with graduate students Kyung Duk Koh and Sathya Balachander \u2013 and at the University of Colorado Anschutz Medical School with assistant professor Jay Hesselberth.\u003C\/p\u003E\u003Cp\u003EThe research was supported by the National Science Foundation, the Georgia Research Alliance, the American Cancer Society, the Damon Runyon Cancer Research Foundation, and the University of Colorado Golfers Against Cancer.\u003C\/p\u003E\u003Cp\u003EBecause of the extra hydroxyl (OH) group in the ribonucleotides, their presence distorts the DNA and creates sensitive sites where reactions with other molecules can take place. Of particular interest are reactions between the OH and alkaline solutions, which can make the DNA more susceptible to cleavage.\u003C\/p\u003E\u003Cp\u003ERibose-seq takes advantage of this reaction with the hydroxyl group to launch the process of identifying the genomic spectrum of ribonucleotide (rNMP) incorporation. Researchers first cleave the DNA samples at the ribonucleotides, then take the resulting fragments through a specialized process that concludes with generation of a library of DNA sequences that contain the sites of ribonucleotide incorporation and their upstream sequence. High-throughput sequencing of the library and alignment of sequencing reads to a reference genome identifies the profile of rNMP incorporation events.\u003C\/p\u003E\u003Cp\u003E\u201cRibose-seq is specific to directly capturing ribonucleotides embedded in DNA and does not capture RNA primers or Okazaki fragments formed during DNA replication, breaks or abasic sites in DNA,\u201d Storici noted.\u003C\/p\u003E\u003Cp\u003E\u201cFor this reason, ribose-seq has application for rNMP mapping in any genomic DNA, from large nuclear genomes to small genomic molecules such as plasmids and mitochondrial DNA, with no need of standardization procedures,\u201d she said. \u201cIt also allows mapping rNMPs even in conditions in which the DNA is exposed to environmental stressors that damage the DNA by generating breaks and\/or abasic sites.\u201d\u003C\/p\u003E\u003Cp\u003EThe extra hydroxyl group found in the ribonucleotides is key to the ribose-seq technique, said Koh, the paper\u2019s first author. \u201cThe OH group is specific to the ribonucleotides,\u201d he explained. \u201cThat allowed us to build a new tool for recognizing specifically where the ribonucleotides are located.\u201d\u003C\/p\u003E\u003Cp\u003EThe high-throughput sequencing and initial data analysis were done in the Hesselberth laboratory in the Department of Biochemistry and Molecular Genetics at the University of Colorado Anschutz Medical School.\u003C\/p\u003E\u003Cp\u003ETo validate their method, the researchers tested ribose-seq on the much-studied yeast species. The analyses revealed a strong preference for the cytidine and guanosine bases at the ribonucleotide sites.\u003C\/p\u003E\u003Cp\u003E\u201cThe ribonucleotides are not randomly distributed, and there is some preference for specific base sequences and specific base composition of the ribonucleotide itself,\u201d said Koh. \u201cBy looking at the non-random distribution, we found several hotspots in which the ribonucleotides are incorporated into the genome.\u201d\u003C\/p\u003E\u003Cp\u003EKnowledge of where the ribonucleotides cluster could help identify areas of greatest potential for genome instability and lead to a better understanding of how they affect the properties and activities of DNA.\u003C\/p\u003E\u003Cp\u003E\u201cThe fact that we see biases in the base compositions of the ribonucleotides allows us to tell which base is more likely to be incorporated into the DNA,\u201d Koh explained. \u201cIf there are specific signatures of genomic instability that are caused by the ribonucleotides, this will allow us to narrow down the locations and know where they are more likely to be found.\u201d\u003C\/p\u003E\u003Cp\u003EA next step will be to test ribose-seq on other DNA, Koh said. \u201cOur technique could potentially be applied to any genome of any cell type from any organism as long as genomic DNA can be extracted from it,\u201d he added. \u201cIt is independent of specific organisms.\u201d\u003C\/p\u003E\u003Cp\u003EBeyond repair and replication processes, ribonucleotides can also be created in DNA as a result of damage caused by drugs, environmental stressors and other factors. The ribose-seq method could also allow scientists to study the impact of these processes.\u003C\/p\u003E\u003Cp\u003E\u201cRibose-seq should allow us to better understand the impact of ribonucleotides on the structure and function of DNA,\u201d said Storici. \u201cIdentifying specific signatures of ribonucleotide incorporation in DNA may represent novel biomarkers for human diseases such as cancer, and other degenerative disorders.\u201d\u003C\/p\u003E\u003Cp\u003E\u003Cem\u003EThis material is based upon work supported by the National Science Foundation (NSF) under grant number MCB-1021763, by the Georgia Research Alliance under award number R9028, by the American Cancer Society, by the Damon Runyon Cancer Research Foundation and by the University of Colorado Golfers Against Cancer. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the sponsoring agencies.\u003C\/em\u003E\u003C\/p\u003E\u003Cp\u003E\u003Cstrong\u003ECITATION\u003C\/strong\u003E: Kyung Duk Koh, Sathya Balachander, Jay Hesselberth and Francesca Storici, \u201cRibose-seq: global mapping of ribonucleotides embedded in genomic DNA,\u201d (Nature Methods, 2015). \u003Ca href=\u0022http:\/\/dx.doi.org\/10.1038\/nmeth.3259\u0022\u003Ehttp:\/\/dx.doi.org\/10.1038\/nmeth.3259\u003C\/a\u003E\u003Cbr \/\u003E\u003Cbr \/\u003E\u003Cstrong\u003EResearch News\u003C\/strong\u003E\u003Cbr \/\u003E\u003Cstrong\u003EGeorgia Institute of Technology\u003C\/strong\u003E\u003Cbr \/\u003E\u003Cstrong\u003E177 North Avenue\u003C\/strong\u003E\u003Cbr \/\u003E\u003Cstrong\u003EAtlanta, Georgia\u0026nbsp; 30332-0181\u003C\/strong\u003E\u003Cbr \/\u003E\u003Cbr \/\u003E\u003Cstrong\u003EMedia Relations Contacts\u003C\/strong\u003E: John Toon (404-894-6986) (\u003Ca href=\u0022mailto:jtoon@gatech.edu\u0022\u003Ejtoon@gatech.edu\u003C\/a\u003E) or Brett Israel (404-385-1933) (\u003Ca href=\u0022mailto:brett.israel@comm.gatech.edu\u0022\u003Ebrett.israel@comm.gatech.edu\u003C\/a\u003E)\u003C\/p\u003E\u003Cp\u003E\u003Cstrong\u003EWriter\u003C\/strong\u003E: John Toon\u003C\/p\u003E","summary":null,"format":"limited_html"}],"field_subtitle":"","field_summary":[{"value":"\u003Cp\u003EResearchers have developed and tested a new technique known as ribose-seq that allows them to determine the full profile of ribonucleotides -- RNA fragments -- embedded in genomic DNA.\u003C\/p\u003E","format":"limited_html"}],"field_summary_sentence":[{"value":"Researchers have developed and tested a new technique that allows them to determine the full profile of ribonucleotides -- RNA fragments -- embedded in genomic DNA."}],"uid":"27303","created_gmt":"2015-01-26 11:29:41","changed_gmt":"2016-10-08 03:17:54","author":"John Toon","boilerplate_text":"","field_publication":"","field_article_url":"","dateline":{"date":"2015-01-26T00:00:00-05:00","iso_date":"2015-01-26T00:00:00-05:00","tz":"America\/New_York"},"extras":[],"hg_media":{"368141":{"id":"368141","type":"image","title":"Ribose-seq identifies ribonucleotides2","body":null,"created":"1449245827","gmt_created":"2015-12-04 16:17:07","changed":"1475895107","gmt_changed":"2016-10-08 02:51:47","alt":"Ribose-seq identifies ribonucleotides2","file":{"fid":"74895","name":"ribonucleotides-in-dna7.jpg","image_path":"\/sites\/default\/files\/images\/ribonucleotides-in-dna7.jpg","image_full_path":"http:\/\/hg.gatech.edu\/\/sites\/default\/files\/images\/ribonucleotides-in-dna7.jpg","mime":"image\/jpeg","size":1865589,"path_740":"http:\/\/hg.gatech.edu\/sites\/default\/files\/styles\/740xx_scale\/public\/images\/ribonucleotides-in-dna7.jpg?itok=7iufMMdJ"}},"368131":{"id":"368131","type":"image","title":"Ribose-seq identifies ribonucleotides","body":null,"created":"1449245827","gmt_created":"2015-12-04 16:17:07","changed":"1475895107","gmt_changed":"2016-10-08 02:51:47","alt":"Ribose-seq identifies ribonucleotides","file":{"fid":"74894","name":"ribonucleotides-in-dna2.jpg","image_path":"\/sites\/default\/files\/images\/ribonucleotides-in-dna2.jpg","image_full_path":"http:\/\/hg.gatech.edu\/\/sites\/default\/files\/images\/ribonucleotides-in-dna2.jpg","mime":"image\/jpeg","size":1473975,"path_740":"http:\/\/hg.gatech.edu\/sites\/default\/files\/styles\/740xx_scale\/public\/images\/ribonucleotides-in-dna2.jpg?itok=iHuwHP7O"}},"368161":{"id":"368161","type":"image","title":"Ribose-seq identifies ribonucleotides3","body":null,"created":"1449245827","gmt_created":"2015-12-04 16:17:07","changed":"1475895107","gmt_changed":"2016-10-08 02:51:47","alt":"Ribose-seq identifies ribonucleotides3","file":{"fid":"74897","name":"ribonucleotides-in-dna6.jpg","image_path":"\/sites\/default\/files\/images\/ribonucleotides-in-dna6.jpg","image_full_path":"http:\/\/hg.gatech.edu\/\/sites\/default\/files\/images\/ribonucleotides-in-dna6.jpg","mime":"image\/jpeg","size":1420336,"path_740":"http:\/\/hg.gatech.edu\/sites\/default\/files\/styles\/740xx_scale\/public\/images\/ribonucleotides-in-dna6.jpg?itok=kql-Zofl"}}},"media_ids":["368141","368131","368161"],"groups":[{"id":"1188","name":"Research Horizons"}],"categories":[{"id":"146","name":"Life Sciences and Biology"},{"id":"135","name":"Research"}],"keywords":[{"id":"116071","name":"DNA. ribonucleotide"},{"id":"116051","name":"Ribose-seq"},{"id":"984","name":"RNA"}],"core_research_areas":[{"id":"39441","name":"Bioengineering and Bioscience"},{"id":"39431","name":"Data Engineering and Science"}],"news_room_topics":[{"id":"71891","name":"Health and Medicine"}],"event_categories":[],"invited_audience":[],"affiliations":[],"classification":[],"areas_of_expertise":[],"news_and_recent_appearances":[],"phone":[],"contact":[{"value":"\u003Cp\u003EJohn Toon\u003C\/p\u003E\u003Cp\u003EResearch News\u003C\/p\u003E\u003Cp\u003E\u003Ca href=\u0022mailto:jtoon@gatech.edu\u0022\u003Ejtoon@gatech.edu\u003C\/a\u003E\u003C\/p\u003E\u003Cp\u003E(404) 894-6986\u003C\/p\u003E","format":"limited_html"}],"email":["jtoon@gatech.edu"],"slides":[],"orientation":[],"userdata":""}}}