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. 2006 Apr;26(8):3327-34.
doi: 10.1128/MCB.26.8.3327-3334.2006.

Replication fork progression is impaired by transcription in hyperrecombinant yeast cells lacking a functional THO complex

Ralf E Wellinger  1 , Félix Prado, Andrés Aguilera
Affiliations

Replication fork progression is impaired by transcription in hyperrecombinant yeast cells lacking a functional THO complex

Ralf E Wellinger et al. Mol Cell Biol. 2006 Apr.

Abstract

THO/TREX is a conserved, eukaryotic protein complex operating at the interface between transcription and messenger ribonucleoprotein (mRNP) metabolism. THO mutations impair transcription and lead to increased transcription-associated recombination (TAR). These phenotypes are dependent on the nascent mRNA; however, the molecular mechanism by which impaired mRNP biogenesis triggers recombination in THO/TREX mutants is unknown. In this study, we provide evidence that deficient mRNP biogenesis causes slowdown or pausing of the replication fork in hpr1Delta mutants. Impaired replication appears to depend on sequence-specific features since it was observed upon activation of lacZ but not leu2 transcription. Replication fork progression could be partially restored by hammerhead ribozyme-guided self-cleavage of the nascent mRNA. Additionally, hpr1Delta increased the number of S-phase but not G(2)-dependent TAR events as well as the number of budded cells containing Rad52 repair foci. Our results link transcription-dependent genomic instability in THO mutants with impaired replication fork progression, suggesting a molecular basis for a connection between inefficient mRNP biogenesis and genetic instability.

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Figures

FIG. 1.
FIG. 1.
Impaired replication fork progression in hpr1Δ mutants. (A) Scheme of the 6.26-kb pRWY005 yeast plasmid constructed for this study (left) and a 2D gel pattern of predictable replication intermediates upon plasmid linearization (right). Depicted are restriction sites, functional elements, and a 2D migration pattern of different forms of replication intermediates or uncut plasmid. nc, nicked circular; Dim., dimension. (B) 2D gel analysis of PmlI-digested plasmid DNA from wild-type (WT) and hpr1::KAN cells. Note that the gel slice used after the first dimension was mainly devoid of the 3-kb nonreplicating SmaI-SacI fragment. The transcriptional status of lacZ from cells grown either in galactose (ON) or in glucose (OFF) is indicated. The arrow points to a very faint signal corresponding to bubble-shaped molecules derived from hpr1Δ cells grown in galactose. Note that a weak signal intensity for the bubble arc was repeatedly obtained with PmlI-digested DNA. (C) Scheme of fork progression in galactose based on the replication intermediates shown in panel B. The switch from bubble- to double-Y-shaped molecules (arrow) and the region of replication termination (gray bar) are indicated. Representative gels of two independent experiments are shown.
FIG. 2.
FIG. 2.
Slowdown of replication fork progression at the 3′ end of the transcribed lacZ gene. (A) 2D gel migration pattern of replication intermediates of the 3-kb SmaI-SacI fragment. Schemes of the expected replication intermediates (left) in which the predicted transition from bubble to simple-Y arc (dashed line) and the migration pattern after 2D gel electrophoresis (right) are indicated. Depicted are the locations of simple-Y-shaped replication intermediates corresponding to normally progressing (R1) or impaired (R2) replication forks. (B) 2D gel analysis of replication intermediates isolated from wild-type (WT) or hpr1::KAN cells grown in glucose (top) and galactose (bottom). The profile of replication intermediate distribution within the descending simple-Y arc is represented, and the quantification data of the regions corresponding to R1- or R2-type molecules are presented underneath the gels. Similar numbers were obtained from analysis of replication intermediates derived from two independent experiments. Note that the intense spot signal along the arc of linear molecules at the left end of the Y arc may represent unspecific hybridization or single-cut, linearized plasmid, while the spot signal to the right of the left end of the Y arc results from unspecific hybridization (asterisk).
FIG. 3.
FIG. 3.
Ribozyme-mediated self-cleavage of nascent mRNA results in a partial suppression of replication slowdown. (A) Scheme of the region containing the ribozyme sequence. The 346-bp ribozyme is depicted in the form of a secondary-structure RNA sequence placed at the 3′ end of the lacZ transcript. (B) Northern blot analysis of full-length lacZ mRNA transcripts. (C) 2D gel analysis of replication intermediates of pRWY012 constructs isolated from hpr1Δ cells. Hybridization against a 3.2-kb SmaI-SacI fragment is shown. Schemes of the construct containing the inactive (ribm) or active (Rib+) ribozyme sequence are indicated on the top. Distribution and quantification of replication intermediates within the descending simple-Y arc are presented at the bottom. For other details, see the legend to Fig. 2.
FIG. 4.
FIG. 4.
leu2 sequences are not sufficient to induce hpr1Δ-dependent replication constraints. (A) Scheme of a 3-kb fragment containing the leu2 sequence. (B) 2D gel analysis of replication intermediates. Distribution and quantification of replication intermediates within the descending simple-Y arc are presented at the bottom. For other details, see the legend to Fig. 2.
FIG. 5.
FIG. 5.
hpr1::KAN leads to an accumulation of Rad52 foci during replication. Percentages of cells containing Rad52-YFP foci are shown. The average number of foci, as determined in exponentially growing cell cultures obtained from three independent transformants, is shown. Unbudded (G1) and budded (S/G2) cells were counted separately. WT, wild type.
FIG. 6.
FIG. 6.
hpr1Δ-dependent hyperrecombination requires transcription during S phase. Recombination frequencies of leu2 repeat constructs in which transcription of the GAL1 promoter is repressed in 2% glucose (OFF), driven by the S-phase-specific HHF2 (S) or G2-phase-specific CLB2 (G2) promoters in wild-type (WT) and hpr1::KAN cells. (A) Recombination in the leu2Δ5′::leu2Δ5′ repeat construct without an intervening DNA sequence. (B) Recombination in the leu2Δ5′::lacZ::leu2Δ5′ repeat construct containing lacZ between the leu2 repeats. A scheme of each construct is shown on top of each panel. The average and standard deviation of four median frequencies, each obtained from six independent cultures, are indicated for each genotype.
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References

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