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. 1997 Dec 15;11(24):3459-70.
doi: 10.1101/gad.11.24.3459.

The yeast HPR1 gene has a functional role in transcriptional elongation that uncovers a novel source of genome instability

S Chávez  1 , A Aguilera
Affiliations

The yeast HPR1 gene has a functional role in transcriptional elongation that uncovers a novel source of genome instability

S Chávez et al. Genes Dev. .

Abstract

The yeast HPR1 gene plays an important role in genome stability, as indicated by the observation that hpr1 mutants have high frequencies of DNA repeat recombination and chromosome loss. Here we report that HPR1 is required for transcriptional elongation. Transcription driven from constitutive and regulated yeast promoters cannot elongate through the bacterial lacZ coding region in hpr1Delta cells, but progresses efficiently through other sequences such as yeast PHO5. We show that HPR1 is not required for transcription activation and that the previously reported effects of hpr1Delta on the activation of different promoters is a consequence of the incapacity of hpr1Delta cells to elongate transcription through lacZ, used as reporter. Transcriptional defects are also observed in yeast DNA sequences of hpr1Delta cells in the presence of the transcription elongation inhibitor 6-azauracil. In all cases, the blockage of transcription elongation in hpr1Delta is associated with both the high frequency of deletions and the increase in plasmid instability that we report here. Therefore, in addition to the identification of a new element involved in transcriptional elongation, our work provides evidence for a new source of genomic instability.

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Figures

Figure 1
Figure 1
Expression of lacZ fusion constructs placed in CEN plasmids in wild-type and hpr1Δ cells. The hpr1Δ mutation abolished transcription through the bacterial lacZ coding region, regardless of the yeast promoter from which it was transcribed, whereas it did not affect transcription through the yeast PHO5 coding region. (A) The lacZ coding region was fused to either the regulated GAL1 promoter or the constitutive TEF2 and ADH1 promoters. The PHO5 coding region was fused to the GAL1 promoter. Numbers below each construct refer to the translation start of each DNA sequence. Expression of lacZ and PHO5 was determined by β-galactosidase (B–D) or acid phosphatase (E) activities, respectively. For the GAL1-driven expression of either lacZ (B) or PHO5 (C), either 2% glucose (Glu, shaded bars, B,C,E) or 2% galactose (Gal, open bars, B,C,E) was added to 16-hr mid-log phase cultures in glycerol–lactate synthetic medium, and enzymatic activities were assayed 8 hr later.
Figure 2
Figure 2
Expression of the chromosomally located lacZ (A) and PHO5 (B) coding sequences under the control of the PHO5 promoter in wild-type and hpr1Δ cells. For repression (+Pi, shaded bars) and induction (−Pi, open bars) conditions see Materials and Methods. Acid phosphatase and β-galactosidase were assayed in the same culture for each sample. Other details as in Fig. 1.
Figure 3
Figure 3
Northern analysis of GAL1–lacZ and GAL1–PHO5. The Northern analysis and kinetics of induction of lacZ (A,B) and PHO5 (C,D) mRNAs driven from the GAL1 promoter is shown. Wild-type (○, B,D) and hpr1Δ (□, B,D) transformants were obtained from overnight cultures in glycerol–lactate synthetic media lacking uracil and diluted in identical fresh media to an OD600 of 0.5. Galactose (Gal) was then added and samples were taken for Northern analysis after different times, as specified. For repression conditions (Glu) total RNA was isolated from mid-log phase cultures in 2% glucose synthetic media lacking uracil. The DNA probes used were (lacZ 5′ end) the 0.5-kb BamHI–HpaI fragment of pLGZ containing the 5′ end of lacZ; (lacZ 3′ end) the 0.4-kb PvuII fragment of pLGZ containing the 5′- end of lacZ; (PHO5) the 1.5-kb EcoRI–PstI internal PHO5 fragment of pJDB207–PHO5 (Eco); (ACT1) the 0.6-kb ClaI internal ACT1 fragment of plasmid pYA301. (AU) arbitrary units.
Figure 4
Figure 4
Transcriptional run-on analysis in wild-type and hpr1Δ cells. Total RNA was isolated from wild-type and hpr1Δ cells transformed with single-copy p416GAL1–lacZ plasmid under induction (A) and repression (B) conditions. Two percent-galactose or 2% glucose was added to yeast cultures in glycerol–lactate synthetic medium at an OD600 of 0.05, 5 hr prior to the run-on analysis. The 0.6-kb internal ACT1 fragment and seven different DNA fragments (1–7) from the lacZ coding region were immobilized in hybond-N+ filters. The lacZ region covering each of the seven DNA fragments used is shown at the bottom. In all cases, the percentage of radiolabeled mRNA bound to each lacZ fragment was normalized with respect to their corresponding levels in galactose-grown wild-type cells, taken as 100% for each. The orientation of lacZ arrows indicates the direction of transcription. As negative control, we used DNA from Salmonella typhimurium (not shown).
Figure 5
Figure 5
(A) Northern analysis of PHO5 mRNA. The DNA probe used for Northern analysis was the 1.5-kb EcoRI–PstI internal PHO5 fragment of pJDB207–PHO5 (Eco). Arbitrary units of mRNA were calculated according to the same standards for all experiments. (B) The mRNA values are given with respect to rRNA levels (see Materials and Methods). (○) Wild type; (□) hpr1Δ. Other details as in Fig. 3.
Figure 6
Figure 6
Transcription and recombination analysis of direct repeat systems carrying lacZ (3 kb) or PHO5 (1.5 kb) coding regions. A scheme of the deletion product formed by recombination between the direct repeats used is shown (A). The diagram of each direct repeat system indicates the 0.6-kb repeated sequences (shaded boxes), the orientation of the lacZ and PHO5 coding regions, the LEU2 promoter (Prm) and transcription terminators (Ter), and the transcripts driven from the LEU2 promoter (arrow), whose 3′ ends have been made to coincide with the position of the corresponding band in each gel (B). Total RNA was isolated from overnight cultures in synthetic media lacking tryptophan. The DNA probes used in the hybridization experiments were the 598-bp ClaI–EcoRV LEU2 repeat, and the 581-bp ClaI internal ACT1 fragment. The transcript corresponding to the LEU2 endogenous chromosomal band is indicated. No transcript initiates in the internal lacZ and PHO5 internal sequences as determined with specific lacZ and PHO5 DNA probes (data not shown).
Figure 7
Figure 7
Transcription analysis of a yeast ORF in the presence of 6AU. Northern analysis (A) and kinetics of induction (B) of PHO5 mRNAs driven from the GAL1 promoter in the presence of 6AU with and without guanine are shown. The URA3+ wild-type and hpr1Δ strains transformed with pSCh202 were obtained from overnight cultures in glycerol–lactate synthetic-complete media lacking tryptophan and uracil, and diluted in identical fresh media to an OD600 of 0.5 with 100 μg/ml of 6AU with and without 100 μg/ml of guanine. Galactose was added after 2 hr, and samples were taken for Northern analysis after different times, as specified. The mRNA values are given with respect to rRNA levels (B) (see Materials and Methods). Other details as in Fig. 3.
Figure 8
Figure 8
Recombination in the presence of 6AU. Recombination analysis of the direct repeat constructs L–lacZ and L–PHO5 carrying the PHO5 ORF in both possible orientations between the leu2 repeats (see Fig. 6). Recombination frequencies were determined in the URA3+ wild-type (open bar) and hpr1Δ (shaded bar) strains transformed with the appropriate plasmids, grown in media with or without 100 μg/ml of 6AU.
Figure 9
Figure 9
Plasmid instability in the presence of 6AU. Yeast colonies of URA3+ wild-type and hpr1Δ strains transformed with centromeric plasmid pRS314-LA growing on SC − Trp with or without 100 μg/ml of 6AU and with or without 100 μg/ml of guanine.
Figure 10
Figure 10
Alternative models to explain induction of genomic instability by a transcriptional elongation block. An elongating RNA Pol II (a) would be blocked at particular DNA sequences in the absence of Hpr1p in a region located between direct repeats DR and DR′ (b). The RNA Pol II–DNA complex may facilitate DNA breaks, whether or not mediated by a nuclease (c) or may impede progression of the replication fork (d). The collapsed replication fork could eventually facilitate the break of the template strand leading to a double strand break (e), although this step might not be necessary. From either step (c–e), and presumably after exonuclease digestion of one DNA strand, strand pairing between DR′ and DR (f) facilitated by either single-strand annealing, one-ended invasion, or DNA polymerase strand slippage would cause a deletion event and the loss of the intervening region (lost) (g), explaining the hyper-recombination phenotype of hpr1Δ. Otherwise the DNA molecule, either a plasmid or a chromosome, would be lost, explaining the high levels of chromosome and plasmid loss of hpr1Δ cells.
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