The faithful and efficient transcription of genomic DNA into mRNA is crucial for cell survival under DNA damage caused by UV irradiation, oxidative stress or chemical DNA modifications. Similar to arrest of Pol II after making transcription error, DNA lesions can interfere with replication, potentially causing mutations in DNA, and also hinder transcription, affecting genome stability and regulation of gene expression. To maintain genomic integrity, cells have evolved separate cellular strategies involving multiple DNA damage repair and DNA damage tolerance mechanisms. Non-bulky DNA lesions are preferentially repaired by the base excision repair (BER) pathway while damages that cause large DNA distortion, such as UV light-induced cyclobutane pyrimidine dimers (CPDs)/cisplatin adducts and oxidative cyclopurines are primarily subject to the nucleotide excision repair (NER) pathway. Despite ongoing repair, some lesions escape detection, presenting the cell with a challenge for continued DNA and RNA synthesis. During replication, the deleterious effect of DNA lesions can be alleviated by translesion DNA synthesis [TLS]. During TLS, the high-fidelity replicative DNA polymerases are switched transiently to specialized translesion DNA polymerases that can accommodate bulky lesions within a more spacious active site, thus enabling their bypass. Recently, we demonstrated that yeast Pol II employs a distinct mechanism for CPD bypass (called A-rule) involving a conformational flexibility of its active center (the mobile trigger loop domain in Rpb1 subunit) facilitating accommodation of bulky lesions. We recently expanded this project to Pol II bypass of bulky cyclopurine (CyPn) oxidative damages in transcribed genes and to analysis of RTLS (RNA polymerase translesion synthesis) through the bulky lesions by mammalian (calf thymus, CT) Pol II. We also plan to identify and test in vitro protein factors involved in RTLS and in initiation of TCR in yeast and mammalian cells. We have already found that mammalian TFIIF significantly promotes RTLS by CT Pol II in vitro.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC011202-06
Application #
8938005
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
6
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
KIreeva, Maria; Trang, Cyndi; Matevosyan, Gayane et al. (2018) RNA-DNA and DNA-DNA base-pairing at the upstream edge of the transcription bubble regulate translocation of RNA polymerase and transcription rate. Nucleic Acids Res 46:5764-5775
Kireeva, Maria L; Afonin, Kirill A; Shapiro, Bruce A et al. (2017) Cotranscriptional Production of Chemically Modified RNA Nanoparticles. Methods Mol Biol 1632:91-105
Bubunenko, Mikhail G; Court, Carolyn B; Rattray, Alison J et al. (2017) A Cre Transcription Fidelity Reporter Identifies GreA as a Major RNA Proofreading Factor in Escherichia coli. Genetics 206:179-187
Herrera-Asmat, Omar; Lubkowska, Lucyna; Kashlev, Mikhail et al. (2017) Production and characterization of a highly pure RNA polymerase holoenzyme from Mycobacterium tuberculosis. Protein Expr Purif 134:1-10
Walmacq, Celine; Wang, Lanfeng; Chong, Jenny et al. (2015) Mechanism of RNA polymerase II bypass of oxidative cyclopurine DNA lesions. Proc Natl Acad Sci U S A 112:E410-9
Sun, Bo; Pandey, Manjula; Inman, James T et al. (2015) T7 replisome directly overcomes DNA damage. Nat Commun 6:10260
Imashimizu, Masahiko; Shimamoto, Nobuo; Oshima, Taku et al. (2014) Transcription elongation: Heterogeneous tracking of RNA polymerase and its biological implications. Transcription 5:
Imashimizu, Masahiko; Shimamoto, Nobuo; Oshima, Taku et al. (2014) Transcription elongation. Heterogeneous tracking of RNA polymerase and its biological implications. Transcription 5:e28285
Walmacq, Celine; Cheung, Alan C M; Kireeva, Maria L et al. (2012) Mechanism of translesion transcription by RNA polymerase II and its role in cellular resistance to DNA damage. Mol Cell 46:18-29
Walmacq, Celine; Kireeva, Maria L; Irvin, Jordan et al. (2009) Rpb9 subunit controls transcription fidelity by delaying NTP sequestration in RNA polymerase II. J Biol Chem 284:19601-12