Guanine repeat-containing sequences confer transcription-dependent instability in an orientation-specific manner in yeast

Abstract

Non-B DNA structures are a major contributor to the genomic instability associated with repetitive sequences. Immunoglobulin switch Mu (Sμ) region sequence is comprised of guanine-rich repeats and has high potential for forming G4 DNA, in which one strand of DNA folds into an array of guanine quartets. Taking advantage of the genetic tractability of Saccharomyces cerevisiae, we developed a recombination assay to investigate mechanisms involved in maintaining stability of G-rich repetitive sequence. By embedding Sμ sequence within recombination substrates under the control of a tetracycline-regulatable promoter, we demonstrate that the rate and orientation of transcription both affect the stability of Sμ sequence. In particular, the greatest instability was observed under high-transcription conditions when the Sμ sequence was oriented with the C-rich strand as the transcription template. The effect of transcription orientation was enhanced in the absence of the Type IB topoisomerase Top1, possibly due to enhanced R-loop formation. Loss of Sgs1 helicase and RNase H activity also increased instability, suggesting they may cooperatively function to reduce the formation of non-B DNA structures in highly transcribed regions. Finally, the Sμ sequence was unstable when transcription elongation was perturbed due to a defective THO complex. In a THO-deficient background, there was further exacerbation of orientation-dependent instability associated with the ectopically expressed, single-strand cytosine deaminase AID. The implications of our findings to understanding instability associated with potential G4 DNA forming sequences are discussed.

Figure 1

System for monitoring Sμ-associated recombination. A. Sequence of the DpnI fragment of murine immunoglobulin Sμ region inserted into the BglII site of LYS2 gene. Uninterrupted (GAGCT)nGGGGT repeat units are indicated in bold italics and 4G runs are underlined. B. Transcription orientations in the pTET-lys2-SμF and pTET-lys2-SμR constructs. Transcription bubbles are illustrated with the gray line indicating nascent mRNA. The single-stranded, non-transcribed strand is G-rich in the pTET-lys2-SμF construct and C-rich in the pTET-lys2-SμR. C. Chromosomal configurations of the recombination substrates (see Material and Methods for details). Black and white circles indicate the locations of centromeres relative to the lys2 alleles on Chr. III and Chr. XV, respectively. Recombination initiated by a gap or break within pTET-lys2 allele removes the insert to create a LYS⁺ pTET-LYS2 allele. Removal of the insert by recombination associated with a crossover event result in unviable acentric and dicentric products.

Figure 2

Regulation of G-loop formation in highly transcribed DNA. See Discussion for details.