DNA Instability Maintains the Repeat Length of the Yeast RNA Polymerase II C-terminal Domain

Abstract

The C-terminal domain (CTD) of RNA polymerase II in eukaryotes is comprised of tandemly repeating units of a conserved seven-amino acid sequence. The number of repeats is, however, quite variable across different organisms. Furthermore, previous studies have identified evidence of rearrangements within the CTD coding region, suggesting that DNA instability may play a role in regulating or maintaining CTD repeat number. The work described here establishes a clear connection between DNA instability and CTD repeat number in Saccharomyces cerevisiae. First, analysis of 36 diverse S. cerevisiae isolates revealed evidence of numerous past rearrangements within the DNA sequence that encodes the CTD. Interestingly, the total number of CTD repeats was relatively static (24–26 repeats in all strains), suggesting a balancing act between repeat expansion and contraction. In an effort to explore the genetic plasticity within this region, we measured the rates of repeat expansion and contraction using novel reporters and a doxycycline-regulated expression system for RPB1. In efforts to determine the mechanisms leading to CTD repeat variability, we identified the presence of DNA secondary structures, specifically G-quadruplex-like DNA, within the CTD coding region. Furthermore, we demonstrated that mutating PIF1, a G-quadruplex-specific helicase, results in increased CTD repeat length polymorphisms. We also determined that RAD52 is necessary for CTD repeat expansion but not contraction, identifying a role for recombination in repeat expansion. Results from these DNA rearrangements may help explain the CTD copy number variation seen across eukaryotes, as well as support a model of CTD expansion and contraction to maintain CTD integrity and overall length.

Comprehensive RNA Polymerase II Interactomes Reveal Distinct and Varied Roles for Each Phospho-CTD Residue

“…A less stringent and inducible T4V mutant was created by generating an allele of RPB1 that encodes a CTD where Thr4 of the first 8 repeats proximal to the Pol II core are mutated to valine (T4V 1–8 ). By placing the endogenous RPB1 under control of a Tet-off promoter, introduction of doxycycline induces incorporation of the mutant CTD into the Pol II complex (Malagon et al, 2006; Morrill et al, 2016.). NET-seq analysis in this T4V 1–8 mutant shows defects similar to the T4V 1–26 mutant in post-polyA transcription (Figure S3B).…”

Template Switching Mediates Contractions of a Repetitive Coding Region inS. cerevisiae

“…Doxycycline (+DOX, 50 µg/ml) was added to plates to control the expression of genomic RPB1 when appropriate. Plasmids used in this work were described previously in Morrill et al 2016.…”

“…While the mechanisms of repeat instability in simple repeat sequences, such as trinucleotide repeats, are well studied (8,19), little is known about how more complex repeat sequences like the CTD promote variability. Our lab previously determined that expansions of the CTD require Rad52p, suggesting that expansions result from homologous recombination (4). In this work, we sought to determine the mechanism(s) by which the CTD contracts.…”

“…We previously reported the mutation rate for cells expressing a plasmid-based mutant CTD construct designed to monitor contractions, p4stop (4). This construct encodes all 26 yeast CTD repeats, with repeats 8-11 each containing a stop codon (Tyr1àstop) and a non-coding Ser2àTrp mutation ( Figure 2A).…”

“…Despite being an essential region of an essential gene, we have demonstrated that the CTD coding region is genetically unstable. In addition to repeat length variation across organisms, previous work by our lab has shown that the CTD varies in length somewhat across strains of yeast, and yeast with a suboptimal CTD length or mutated CTD sequence are capable of promoting spontaneous expansion or contraction of the coding region in order to improve fitness (4). Repeat instability may therefore serve as a mechanism for reducing mutagenesis in an essential sequence by promoting the removal or templated repair of damaged repeats while maintaining overall length.…”