Book of Abstracts: Albany 2011

category image Albany 2011
Conversation 17
June 14-18 2011
©Adenine Press (2010)

Transcription-associated Mutagenesis and Top1 Activity in Budding Yeast

High levels of transcription stimulate both homologous recombination and mutagenesis in yeast, and do so by multiple mechanisms (1). Using the CAN1 gene fused to the GAL1 promoter, a novel, 2-5 bp deletion signature associated with high levels of transcription has been identified. These deletions accumulate at discrete hotspots that coincide with short tandem repeats of the same size, and are completely dependent on the activity of Top1 (2, 3). Top1 is a type 1B topoisomerase that nicks DNA, forming a reversible intermediate with the enzyme covalently attached at the nick via a 3’ phosphotyrosyl linkage (4). We propose that the deletions reflect the processing of a Top1 cleavage product generated during the removal of transcription-associated supercoils. To study the genetic control of deletion formation, individual 2-bp deletions hotspots have been transplanted into a frameshift reversion assay where they can be studied in isolation. In this much more sensitive system, Top1-dependent deletions are observed even under low-transcription conditions. Because of the similarity of the Top1-dependent deletion signature to that recently associated with a failure to remove ribonucleotides (rNMPs) from genomic DNA (an RNase H2-deficient background; ref. 5), we have explored whether there is any relationship between the two. We find that rNMP-initiated deletions that accumulate in the absence of RNase H2 require the activity of Top1. This suggests a model in which presence of an rNMP at the scissile phosphate leads to an irreversible Top1 cleavage product (6) that is subsequently processed into a deletion intermediate. While all rNMP-initiated deletions appear to require Top1, there are subclasses of Top1-dependent deletions that are not elevated in the absence of RNase H2. We propose that rNMP-independent deletions reflect processing of a reversible Top1 cleavage intermediate that becomes trapped on DNA. Finally, we demonstrate that there is a synergistic relationship between rNMP-associated deletions and high levels of transcription, suggesting that rNMP levels within genomic DNA can be influenced by transcriptional status.

This work has been supported by NIH grants GM38464 and GM93197 awarded to SJR.

  1. A. Aguilera, B. Gomez-Gonzalez, Nat Rev Genet 9, 204-217 (2008).
  2. M. J. Lippert et al., Proc Natl Acad Sci USA 108, 698-703 (2011).
  3. T. Takahashi, G. Burguiere-Slezak, P. A. Van der Kemp, S. Boiteux, Proc Natl Acad Sci USA 108, 692-697 (2011).
  4. J. C. Wang, Nat Rev Mol Cell Biol 3, 430-440 (2002).
  5. S. A. McElhinny et al., Nat Chem Biol 6, 774-781 (2010).
  6. J. Sekiguchi, S. Shuman, Mol Cell 1, 89-97 (1997).

Nayun Kim
Jang-Eun Cho
Sue Jinks-Robertson

Department of Molecular Genetics and Microbiology
Duke University Medical Center
Durham, NC 27710 USA

Ph: (919) 681-7273
Fx: (919) 684-2790