RNA-POLYMERASE BYPASS AT SITES OF DIHYDROURACIL - IMPLICATIONS FOR TRANSCRIPTIONAL MUTAGENESIS

Dihydrouracil (DHU) is a major base damage product formed from cytosin e following exposure of DNA to ionizing radiation under anoxic conditi ons. To gain insight into the DNA lesion structural requirements for R NA polymerase arrest or bypass at various DNA damages located on the t ranscribed strand during elongation, DHU was placed onto promoter-cont aining DNA templates 20 nucleotides downstream from the transcription start site. In vitro, single-round transcription experiments carried o ut with SP6 and T7 RNA polymerases revealed that following a brief pau se at the DHU site, both enzymes efficiently bypass this lesion with s ubsequent rapid generation of full-length runoff transcripts. Direct s equence analysis of these transcripts indicated that both RNA polymera ses insert primarily adenine opposite to the DHU site, resulting in a G-to-A transition mutation in the lesion bypass product, Such bypass a nd insertion events at DHU sites (or other types of DNA damages), if t hey occur in vivo, have a number of important implications for both th e repair of such lesions and the DNA damage-induced production of muta nt proteins at the level of transcription (transcriptional mutagenesis ).