The fidelity of transcription in human cells.

To determine the error rate of transcription in human cells, we analyzed the transcriptome of H1 human embryonic stem cells with a circle-sequencing approach that allows for high-fidelity sequencing of the transcriptome. These experiments identified approximately 100,000 errors distributed over every major RNA species in human cells. Our results indicate that different RNA species display different error rates, suggesting that human cells prioritize the fidelity of some RNAs over others.

A Transcription Fidelity Reporter Identifies GreA as a Major RNA Proofreading Factor in .

We made a coupled genetic reporter that detects rare transcription misincorporation errors to measure RNA polymerase transcription fidelity in Using this reporter, we demonstrated that the transcript cleavage factor GreA, but not GreB, is essential for proofreading of a transcription error where a riboA has been misincorporated instead of a riboG. A mutant strain had more than a 100-fold increase in transcription errors relative to wild-type or a mutant. However, overexpression of GreB in Δ cells reduced the misincorporation errors to wild-type levels, demonstrating that GreB at high concentration could substitute for GreA in RNA proofreading activity .

RNA polymerase II senses obstruction in the DNA minor groove via a conserved sensor motif.

RNA polymerase II (pol II) encounters numerous barriers during transcription elongation, including DNA strand breaks, DNA lesions, and nucleosomes. Pyrrole-imidazole (Py-Im) polyamides bind to the minor groove of DNA with programmable sequence specificity and high affinity. Previous studies suggest that Py-Im polyamides can prevent transcription factor binding, as well as interfere with pol II transcription elongation.

Transcription errors induce proteotoxic stress and shorten cellular lifespan.

Transcription errors occur in all living cells; however, it is unknown how these errors affect cellular health. To answer this question, we monitor yeast cells that are genetically engineered to display error-prone transcription. We discover that these cells suffer from a profound loss in proteostasis, which sensitizes them to the expression of genes that are associated with protein-folding diseases in humans; thus, transcription errors represent a new molecular mechanism by which cells can acquire disease phenotypes.

Elevated mutation rate during meiosis in Saccharomyces cerevisiae.

Mutations accumulate during all stages of growth, but only germ line mutations contribute to evolution. While meiosis contributes to evolution by reassortment of parental alleles, we show here that the process itself is inherently mutagenic. We have previously shown that the DNA synthesis associated with repair of a double-strand break is about 1000-fold less accurate than S-phase synthesis.

A genetic assay for transcription errors reveals multilayer control of RNA polymerase II fidelity.

We developed a highly sensitive assay to detect transcription errors in vivo. The assay is based on suppression of a missense mutation in the active site tyrosine in the Cre recombinase. Because Cre acts as tetramer, background from translation errors are negligible.

GAP-Seq: a method for identification of DNA palindromes.

Background: Closely spaced long inverted repeats, also known as DNA palindromes, can undergo intrastrand annealing to form DNA hairpins. The ability to form these hairpins results in genome instability, difficulties in maintaining clones in Escherichia coli and major problems for most DNA sequencing approaches. Because of their role in genomic instability and gene amplification in some human cancers, it is important to develop systematic approaches to detect and characterize DNA palindromes.

Intrinsic translocation barrier as an initial step in pausing by RNA polymerase II.

Pausing of RNA polymerase II (RNAP II) by backtracking on DNA is a major regulatory mechanism in control of eukaryotic transcription. Backtracking occurs by extrusion of the 3' end of the RNA from the active center after bond formation and before translocation of RNAP II on DNA. In several documented cases, backtracking requires a special signal such as A/T-rich sequences forming an unstable RNA-DNA hybrid in the elongation complex.

Isolation and characterization of RNA polymerase rpoB mutations that alter transcription slippage during elongation in Escherichia coli.

Transcription fidelity is critical for maintaining the accurate flow of genetic information. The study of transcription fidelity has been limited because the intrinsic error rate of transcription is obscured by the higher error rate of translation, making identification of phenotypes associated with transcription infidelity challenging. Slippage of elongating RNA polymerase (RNAP) on homopolymeric A/T tracts in DNA represents a special type of transcription error leading to disruption of open reading frames in Escherichia coli mRNA.

The fidelity of transcription: RPB1 (RPO21) mutations that increase transcriptional slippage in S. cerevisiae.

The fidelity of RNA synthesis depends on both accurate template-mediated nucleotide selection and proper maintenance of register between template and RNA. Loss of register, or transcriptional slippage, is particularly likely on homopolymeric runs in the template. Transcriptional slippage can alter the coding capacity of mRNAs and is used as a regulatory mechanism.

Mechanism of translesion transcription by RNA polymerase II and its role in cellular resistance to DNA damage.

UV-induced cyclobutane pyrimidine dimers (CPDs) in the template DNA strand stall transcription elongation by RNA polymerase II (Pol II). If the nucleotide excision repair machinery does not promptly remove the CPDs, stalled Pol II creates a roadblock for DNA replication and subsequent rounds of transcription. Here we present evidence that Pol II has an intrinsic capacity for translesion synthesis (TLS) that enables bypass of the CPD with or without repair.

Isolation and characterization of transcription fidelity mutants.

Accurate transcription is an essential step in maintaining genetic information. Error-prone transcription has been proposed to contribute to cancer, aging, adaptive mutagenesis, and mutagenic evolution of retroviruses and retrotransposons. The mechanisms controlling transcription fidelity and the biological consequences of transcription errors are poorly understood.

Rpb9 subunit controls transcription fidelity by delaying NTP sequestration in RNA polymerase II.

Rpb9 is a small non-essential subunit of yeast RNA polymerase II located on the surface on the enzyme. Deletion of the RPB9 gene shows synthetic lethality with the low fidelity rpb1-E1103G mutation localized in the trigger loop, a mobile element of the catalytic Rpb1 subunit, which has been shown to control transcription fidelity. Similar to the rpb1-E1103G mutation, the RPB9 deletion substantially enhances NTP misincorporation and increases the rate of mismatch extension with the next cognate NTP in vitro.

Transient reversal of RNA polymerase II active site closing controls fidelity of transcription elongation.

To study fidelity of RNA polymerase II (Pol II), we analyzed properties of the 6-azauracil-sensitive and TFIIS-dependent E1103G mutant of rbp1 (rpo21), the gene encoding the catalytic subunit of Pol II in Saccharomyces cerevisiae. Using an in vivo retrotransposition-based transcription fidelity assay, we observed that rpb1-E1103G causes a 3-fold increase in transcription errors. This mutant showed a 10-fold decrease in fidelity of transcription elongation in vitro.

Non-homologous end joining is important for repair of Cr(VI)-induced DNA damage in Saccharomyces cerevisiae.

Hexavalent chromium is known to be a potent carcinogen that leads to many different DNA lesions, including DNA-protein crosslinks, and single- and double-strand breaks. In Saccharomyces cerevisiae, DNA double-strand breaks are mainly repaired by either homologous recombination (HR) or non-homologous end-joining (NHEJ) repair pathways. Here, we show that mutants deficient in NHEJ (yku70Delta, rad50Delta, dnl4Delta, mre11Delta, xrs2Delta) of S.

Mutations in the Saccharomyces cerevisiae RPB1 gene conferring hypersensitivity to 6-azauracil.

RNA polymerase II (RNAPII) in eukaryotic cells drives transcription of most messenger RNAs. RNAPII core enzyme is composed of 12 polypeptides where Rpb1 is the largest subunit. To further understand the mechanisms of RNAPII transcription, we isolated and characterized novel point mutants of RPB1 that are sensitive to the nucleotide-depleting drug 6-azauracil (6AU).