title: Mechanism of RNA polymerase II bypass of oxidative cyclopurine DNA lesions.
abstract: In human cells, the oxidative DNA lesion 8,5'-cyclo-2'-deoxyadenosine (CydA) induces prolonged stalling of RNA polymerase II (Pol II) followed by transcriptional bypass, generating both error-free and mutant transcripts with AMP misincorporated immediately downstream from the lesion. Here, we present biochemical and crystallographic evidence for the mechanism of CydA recognition. Pol II stalling results from impaired loading of the template base (5') next to CydA into the active site, leading to preferential AMP misincorporation. Such predominant AMP insertion, which also occurs at an abasic site, is unaffected by the identity of the 5'-templating base, indicating that it derives from nontemplated synthesis according to an A rule known for DNA polymerases and recently identified for Pol II bypass of pyrimidine dimers. Subsequent to AMP misincorporation, Pol II encounters a major translocation block that is slowly overcome. Thus, the translocation block combined with the poor extension of the dA.rA mispair reduce transcriptional mutagenesis. Moreover, increasing the active-site flexibility by mutation in the trigger loop, which increases the ability of Pol II to accommodate the bulky lesion, and addition of transacting factor TFIIF facilitate CydA bypass. Thus, blocking lesion entry to the active site, translesion A rule synthesis, and translocation block are common features of transcription across different bulky DNA lesions.
authors.affiliation: Center for Cancer Research, National Cancer Institute, Frederick, MD 21702;
authors.forename: Celine
authors.initials: C
authors.lastname: Walmacq
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093;
authors.forename: Lanfeng
authors.initials: L
authors.lastname: Wang
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093;
authors.forename: Jenny
authors.initials: J
authors.lastname: Chong
authors.affiliation: Center for Cancer Research, National Cancer Institute, Frederick, MD 21702;
authors.forename: Kathleen
authors.initials: K
authors.lastname: Scibelli
authors.affiliation: Center for Cancer Research, National Cancer Institute, Frederick, MD 21702;
authors.forename: Lucyna
authors.initials: L
authors.lastname: Lubkowska
authors.affiliation: Biopolcore, Inc., Baltimore, MD 21208; and.
authors.forename: Averell
authors.initials: A
authors.lastname: Gnatt
authors.affiliation: Laboratory of Neurogenetics and Division of Metabolism and Health Effects, National Institute on Alcohol Abuse and Alcoholism, and Office of Rare Diseases Research, National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD 20892.
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Brooks
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093; dongwang@ucsd.edu kashlevm@mail.nih.gov.
authors.forename: Dong
authors.initials: D
authors.lastname: Wang
authors.affiliation: Center for Cancer Research, National Cancer Institute, Frederick, MD 21702; dongwang@ucsd.edu kashlevm@mail.nih.gov.
authors.forename: Mikhail
authors.initials: M
authors.lastname: Kashlev
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000737:chemistry; D004249:DNA Damage; D010084:Oxidation-Reduction; D011687:Purines / Q000378:metabolism*; D012319:RNA Polymerase II / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D011687:Purines; D004247:DNA; D012319:RNA Polymerase II
keywords: RNA polymerase II; oxidative DNA damage; transcription factor TFIIF; transcriptional mutagenesis; translesion transcription