Accumulation of single-stranded DNA and destabilization of telomeric repeats in yeast mutant strains carrying a deletion of RAD27 - PubMed (original) (raw)

Accumulation of single-stranded DNA and destabilization of telomeric repeats in yeast mutant strains carrying a deletion of RAD27

J Parenteau et al. Mol Cell Biol. 1999 Jun.

Abstract

The Saccharomyces cerevisiae RAD27 gene encodes the yeast homologue of the mammalian FEN-1 nuclease, a protein that is thought to be involved in the processing of Okazaki fragments during DNA lagging-strand synthesis. One of the predicted DNA lesions occurring in rad27 strains is the presence of single-stranded DNA of the template strand for lagging-strand synthesis. We examined this prediction by analyzing the terminal DNA structures generated during telomere replication in rad27 strains. The lengths of the telomeric repeat tracts were found to be destabilized in rad27 strains, indicating that naturally occurring direct repeats are subject to tract expansions and contractions in such strains. Furthermore, abnormally high levels of single-stranded DNA of the templating strand for lagging-strand synthesis were observed in rad27 cells. Overexpression of Dna2p in wild-type cells also yielded single-stranded DNA regions on telomeric DNA and caused a cell growth arrest phenotype virtually identical to that seen for rad27 cells grown at the restrictive temperature. Furthermore, overexpression of the yeast exonuclease Exo1p alleviated the growth arrest induced by both conditions, overexpression of Dna2p and incubation of rad27 cells at 37 degrees C. However, the telomere heterogeneity and the appearance of single-stranded DNA are not prevented by the overexpression of Exo1p in these strains, suggesting that this nuclease is not simply redundant with Rad27p. Our data thus provide in vivo evidence for the types of DNA lesions predicted to occur when lagging-strand synthesis is deficient and suggest that Dna2p and Rad27p collaborate in the processing of Okazaki fragments.

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Figures

FIG. 1

Nucleotide composition of templating and newly synthesized strands on telomeric repeats. Due to the conserved polarity of the telomeric repeats on all telomeres, the templating strand for lagging-strand synthesis is always the G-rich strand and the templating strand for leading-strand synthesis is the C-rich strand. Thick lines with nucleotides in boldface, templating strands; thin lines, newly synthesized strands. Note that the identity of the last nucleotide at the end of each strand is unknown; the designation drawn out here is given for simplicity.

FIG. 2

Telomeric repeat instability in _rad27_Δ strains. (A) Genomic DNA isolated from RAD27 (WT) or _rad27_Δ cells that were incubated at 23 or 37°C for the indicated times was digested with the restriction endonuclease _Xho_I and analyzed by Southern blotting. The probe used was randomly labeled telomeric repeat DNA. Y′ TRFs migrate at about 1.3 kb, and some of the other bands correspond to non-Y′ TRFs. M, end-labeled 1-kb ladder DNA serving as a size standard. (B) The same blot as in panel A, rehybridized to a probe specific for CEN4 sequences. (C) The same experiment as in panel A, except that the strains were grown at 30°C. Note that _rad27_Δ cells do grow at this temperature. Every day, an aliquot of the cultures was diluted into fresh medium and the cells were regrown to stationary phase (days of culturing are indicated on top of the gel). From cell density measurements, it was calculated that the cultures had grown for approximately 100 generations on day 8 (data not shown).

FIG. 3

Appearance of single-stranded DNA on telomeres derived from _rad27_Δ cells. (A) Genomic DNA isolated from RAD27 (WT) or _rad27_Δ cells incubated at 37°C for the indicated times was digested with _Xho_I and analyzed by a nondenaturing in-gel hybridization procedure (11) with an end-labeled CA oligonucleotide as a probe. Double-stranded and linearized pMW55 served as a negative control (labeled ds), and single-stranded phagemid DNA containing yeast telomeric repeats of the G-rich strands served as a positive control (labeled GT) (see Materials and Methods). Molecular size standards are as in Fig. 2. (B) The DNA in the gel shown in panel A was denatured, and the same gel was rehybridized to the probe to show all the telomeric repeat-containing fragments.

FIG. 4

The single-stranded telomeric repeats observed on TRFs derived from _rad27_Δ cells are specific for the G-rich strand and are terminal extensions. RAD27 (WT) or _rad27_Δ cells were pregrown at 23°C, then incubated overnight at 37°C, and DNA was isolated from both cultures. This DNA was then digested with mung bean nuclease (lanes labeled + for MB) or E. coli exonuclease I (lanes labeled + for Exo) or was mock treated (no enzyme; indicated with a minus sign). All the DNAs were then digested with _Xho_I and analyzed by nondenaturing in-gel hybridization as in Fig. 3. For the designation of the control DNAs, see Fig. 3 and Materials and Methods. (A) The gel was hybridized to the end-labeled GT oligonucleotide. (B) The gel shown in panel A was then rehybridized to the end-labeled CA oligonucleotide. Note that due to the fact that the gel was not denatured between the two probings, both the CA and GT single-stranded controls show positive signals after the second probing. (C) A photograph of the ethidium bromide-stained gel prior to the first hybridization is shown to demonstrate approximately equal loading of DNA in all lanes.

FIG. 5

The single strandedness of the telomeric G-rich strand does not extend into neighboring Y′ sequences. DNAs derived from RAD27 (WT) or _rad27_Δ cells incubated overnight at the indicated temperatures were digested with _Xho_I and analyzed as in Fig. 3 and 4. (A) The probe consisted of an end-labeled Y′ oligonucleotide, detecting the same strand as the G-rich telomeric repeat strand, and the sequences are about 500 bp from the Y′-telomeric repeat sequences boundary. (B) The same gel as in panel A, rehybridized to the telomeric CA probe. Since the gel was not denatured between probings, both single-stranded controls are visible. (C) The gel shown in panels A and B was then denatured and rehybridized to the telomeric CA oligonucleotide. During the repeated hybridizations and washings, some of the smaller DNA fragments (below 1.2 kb) had diffused out of the gel (note the losses of the DNA size standard bands of 1 kb and less). Thus, the signal for the 1.3-kb Y′ TRFs is somewhat weaker than expected for this gel. Controls and DNA size standards are as in Fig. 3 and 4.

FIG. 5

FIG. 6

Overexpression of DNA2 leads to nongrowing cells. WT, nontransformed MW35a cells grown on SC media; WT+pGAL-vector, MW35a cells containing the empty vector and grown on SC-Ura plates; WT+pGAL-DNA2, MW35a cells transformed with a plasmid that contained the DNA2 gene under the control of a galactose-inducible promoter (see Materials and Methods for construction of the plasmids). The plates contained glucose (left) or galactose (right) as a carbon source.

FIG. 7

Induction of DNA2 expression leads to a transient increase of single-stranded telomeric G-rich DNA. MW35a cells containing pGAL-DNA2 were pregrown in SC-Ura media containing glycerol and lactate. Expression of DNA2 was then induced by the addition of galactose, and DNA was prepared from the culture at the indicated times after galactose addition. These DNAs were then digested with _Xho_I and analyzed by nondenaturing in-gel hybridization (left) using an end-labeled CA oligonucleotide as a probe. After denaturation of the DNA, the gel was rehybridized to the same probe as a control (right). Controls and molecular size standards are as in Fig. 2 through 5.

FIG. 8

Overexpression of Exo1p does not prevent the formation of single-stranded DNA in _rad27_Δ cells. RAD27 (WT) or _rad27_Δ cells were transformed with either pRS423 (empty vector) or pE1 (labeled pExo1-2μm), grown in selective media, and incubated overnight at the indicated temperatures. DNA was isolated, digested with _Xho_I, and analyzed as in Fig. 3 through 5. (A) Nondenatured gel probed with the CA oligonucleotide. (B) The DNA in the gel shown in panel A was denatured and rehybridized to the same probe. Controls and molecular size standards are as in Fig. 7.

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Accumulation of single-stranded DNA and destabilization of telomeric repeats in yeast mutant strains carrying a deletion of RAD27 - PubMed (original) (raw)