title: Unexpected DNA loss mediated by the DNA binding activity of ribonuclease A.
abstract: Ribonuclease A (RNase A) is widely used in molecular biology research both for analytical assays and for nucleic acid preparation. The catalytic mechanism of RNase A is well understood and absolutely precludes activity on DNA; however anecdotal reports of DNA degradation by RNase A are not uncommon. Here we describe a mechanism by which RNase A treatment can lead to apparent DNA degradation. This results from the surprising finding that RNase A remains functional in a phenol:chloroform mixture, to our knowledge the only enzyme that survives this highly denaturing solvent environment. Although RNase A does not cleave the DNA backbone it is capable of binding to DNA, forming stable RNase A-DNA complexes that partition to the interphase or organic phase during phenol:chloroform purification. The unexpected survival of the RNase A DNA-binding activity in phenol means that these complexes are not dissolved and a substantial amount of RNase A-bound DNA is permanently removed from the aqueous phase and lost on phase separation. This effect will impact DNA recovery from multiple procedures and is likely to represent a source of sequence bias in genome-wide studies. Our results also indicate that the results of analytical studies performed using RNase A must be considered with care.
authors.affiliation: Epigenetics Programme, The Babraham Institute, Cambridge, United Kingdom.
authors.forename: Federico
authors.initials: F
authors.lastname: Donà
authors.affiliation: Epigenetics Programme, The Babraham Institute, Cambridge, United Kingdom.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Houseley
mesh_terms: D000595:Amino Acid Sequence; D002725:Chloroform / Q000737:chemistry; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D009691:Nucleic Acid Denaturation / Q000235:genetics; D019800:Phenol / Q000633:toxicity; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D004268:DNA-Binding Proteins; D016337:RNA, Catalytic; D019800:Phenol; D002725:Chloroform; D012259:Ribonuclease, Pancreatic