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. Author manuscript; available in PMC: 2017 Nov 8.
Published in final edited form as: Cell Rep. 2016 Nov 8;17(7):1747–1754. doi: 10.1016/j.celrep.2016.10.050

Absence of non-histone protein complexes at natural chromosomal pause sites results in reduced replication pausing in aging yeast cells

Marleny Cabral 1, Xin Cheng 1, Sukhwinder Singh 2, Andreas S Ivessa 1,3
PMCID: PMC5396545  NIHMSID: NIHMS828557  PMID: 27829146

Summary

There is substantial evidence that genomic instability increases during aging. Replication pausing (and stalling) at difficult-to-replicate chromosomal sites may induce genomic instability. Interestingly, in aging yeast cells we observed reduced replication pausing at various natural replication pause sites (RPS) in ribosomal DNA (rDNA) and non-rDNA locations (e.g., silent replication origins and tRNA genes). The reduced pausing occurs independent of the DNA helicase Rrm3p, which facilitates replication past these non-histone protein-complex-bound RPS, and is independent of the deacetylase Sir2p. Conditions of caloric restriction (CR), which extend lifespan, also cause reduced replication pausing at the 5S rDNA and at tRNA genes. In aged and CR-cells, the RPS are less occupied by their specific non-histone protein complexes (e.g., the preinitiation complex TFIIIC), likely because members of these complexes have primarily cytosolic localization. These conditions may lead to reduced replication pausing and may lower replication stress at these sites during aging.

In Brief

Since genomic instability increases during aging, replication pausing/stalling at fragile sites is expected to increase. Cabral et al. now find that pausing/stalling decreases in aging and caloric-restricted budding yeast cells. The authors find that reduced pausing results from the absence of non-histone protein complexes which bind to these sites.

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Introduction

In dividing cells, DNA replication stress might be initiated by the uncontrolled activation of replication origins, impediments to replication movement (e.g., tightly bound DNA-protein complexes and secondary DNA structures), collisions of replication and transcription complexes, various forms of uncontrolled metabolic conditions (e.g., insufficient dNTP pools or histone supplies), and DNA damage (e.g., inter-strand crosslinks) (Branzei and Foiani, 2010; Magdalou et al., 2014). In budding yeast, replication may slow or even stall at up to 1,400 chromosomal sites, which include centromeres, tRNA genes and Ty elements, long-terminal repeats (LTRs), subtelomeric and telomeric regions, silent replication origins, and highly transcribed genes (Azvolinsky et al., 2009; Deshpande and Newlon, 1996; Greenfeder and Newlon, 1992; Ivessa et al., 2003; Ivessa et al., 2002; Ivessa et al., 2000; Lemoine et al., 2005; Makovets et al., 2004; Szilard et al., 2010). Many of these sites were identified in chromosome translocations, since these sites may break more often and exhibit the characteristic features of “fragile sites” (Di Rienzi et al., 2009; Hu et al., 2014; Ivessa et al., 2003; Ivessa et al., 2002; Ivessa et al., 2000; Lemoine et al., 2005; Lindstrom et al., 2011; Raveendranathan et al., 2006; Song et al., 2014; Szilard et al., 2010). Usually non-histone protein complexes occupy these sites thereby causing replication slowing/stalling (Deshpande and Newlon, 1996; Ivessa et al., 2003).

In various species, including yeast, and tissues mutation rates, DNA damage including broken DNA and mitotic recombination rates, chromosome translocations, deletions, and duplications are increasing during aging, which may impact aging (Hu et al., 2014; Lindstrom et al., 2011; Lopez-Otin et al., 2013; Maslov et al., 2013; McMurray and Gottschling, 2003). Aging also alters chromatin. For example, loss of the core histones H3 and H4 leads to shortening of the yeast replicative lifespan (RLS), whereas overexpression of H3 and H4 extends lifespan (Feser et al., 2010; Hu et al., 2014; Pal and Tyler, 2016).

Since numerous forms of DNA damage increase during aging and replication stress might be the result of slowing and/or stalling of replication, we wanted to investigate whether replication pausing/stalling increases at known natural replication pause sites (RPS; e.g., tRNA genes) when yeast cells age.

Results

In replicative aging cells replication pauses/stalls with lower rates at the inactive replication fork barrier, 5S rDNA genes and silent replication origins of ribosomal DNA

Since we needed large numbers of aged wild-type (WT) cells (8 and 20 cell divisions) to analyze replication intermediates by DNA 2D-gels, we used the mother-enrichment-program (MEP; Figures 1A and S1A) (Brewer and Fangman, 1988; Ivessa et al., 2003; Lindstrom and Gottschling, 2009). It is induced by estradiol with the purpose to activate the splice-out of two essential genes (UBC9, CDC20), specifically in the daughter cells, to prevent their propagation. Mother cells undergoing ~8 cell divisions are in the first quarter of their lifespan, cells that have divided ~20 times are close to middle-age. As a control (“young” cells), we grew this strain for the same amount of time in the absence of estradiol (not expressing the MEP).

Figure 1. Reduced pausing at natural replication pause sites (RPS) in ribosomal DNA (rDNA) of aging yeast cells.

Figure 1

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A. The mother-enrichment-program (MEP) to purify old yeast cells (Figure S1A). B. The regions of the analyzed ClaI-rDNA fragment and rDNA probe (StuI-BglII) are indicated. C. Determining the amount of extrachromosomal rDNA circles (ERCs) by DNA 2D gels in young and old WT cells. D. Schema of rDNA replication intermediates (ClaI DNA fragment): BU, bubble arc (i.e., replication initiation); RFB, replication fork barrier; X, termination of rDNA replication; RF, replication forks; RI, recombination intermediates; CF, converging forks. Putative broken replication forks (BF) are indicated (Martin-Parras et al., 1992). Pauses of replication forks at the natural RPS in rDNA are indicated (asterisks). E. Predicted and observed rDNA replication intermediates in aging cells. Right side: 2D gels of ClaI-digested DNA of young and old WT cells. Probe: rDNA (StuI-BglII) F. The amount of each replication intermediate is expressed as ratios (i.e., specific intermediate versus total replication structures (i.e., sum of RF (including pauses and RFB), RI/CF, and BU; see cartoon); “old” is compared to “young”). In this way, we normalized for different amounts of total replication structures. We added up all the intensities of the replication fork pauses (i.e., the in-active RFB (*), the 5S rDNA gene (**) and the silent replication origin (ARS, ***)). Standard errors are displayed.

We analyzed replication intermediates in the repeated ribosomal DNA (rDNA) array, which consists of 100–150 identical 9.1 kbp repeats (Figure 1B) (Brewer and Fangman, 1988; Ivessa, 2013; Ivessa et al., 2000). Extrachromosomal rDNA circles (ERCs) can excise from the array and accumulate in mother cells causing aging (Sinclair and Guarente, 1997). Aged mother cells undergoing ~8 and ~20 cell divisions had a ~8-fold and ~26-fold enrichment of ERCs, respectively, compared to young cells (Figures 1C and S1B) (Ivessa et al., 2000). To analyze replication intermediates by 2D gels we digested the DNA with the restriction enzyme ClaI which generates mainly an about 6 kb fragment from the 9.1 kb rDNA repeat unit (Figures 1B and D) (Ivessa, 2013; Ivessa et al., 2000). rDNA replication is initiated in a subset of the rDNA repeats. The pattern of replication intermediates for any rDNA fragment containing a replication origin (ARS: autonomous replicative sequence) is a composite of intermediates generated from repeats with an active ARS and those without an active ARS (Figure 1B) (Brewer and Fangman, 1988; Ivessa et al., 2000). For repeats with an active ARS, initiation begins and proceeds bi-directionally from the ARS (bubble structures, Figure 1D, BU). They are converted to simple Y structures when the rightward-moving fork reaches the end of the fragment. The leftward-moving fork stops at the site-specific replication fork barrier (RFB) creating increased hybridization on the arc of simple Y-shaped intermediates (Figure 1D, RFB). The remaining part of ClaI fragments with active ARSs is replicated by forks converging on the fork stalled at the RFB. These forks generate intermediates that emanate from the RFB and terminate with a mass of close to 2N (Figure 1D, X). Repeats without an active origin are replicated uni-directionally, creating a full arc of simple Y-shaped intermediates (Figure 1D, passive replication movement).

We investigated whether natural replication pause sites (RPS; e.g., the inactive RFB, 5S rDNA genes, and silent rDNA replication origins) experience increased pausing in aged cells (Figure 1E) (Lindstrom et al., 2011; Vijg and Suh, 2013). Surprisingly, we observed strongly reduced pausing at these sites in old cells except for the active RFB, which was only slightly reduced (Figures 1E, 1F (quantitation), and S1C). The amount of putative rDNA recombination intermediates and converging forks (Figure 1D, RI/CF) was also largely reduced in old cells (Figures 1E and 1F (quantitation)). For example, strains expressing mutated forms of nuclear DNA polymerase α (e.g.: pol12) highly induce the formation of recombination intermediates in rDNA (Zou and Rothstein, 1997).

The reduced replication pausing/stalling at natural RPS in rDNA is independent of Rrm3p and Sir2p, occurs on both chromosomal rDNA and ERCs during aging, and is partly recapitulated under caloric-restricted growth conditions

The DNA helicase Rrm3p is proposed to facilitate replication at over 1,400 chromosomal sites (e.g., tRNA genes, silent replication origins, and sites within the ribosomal DNA (Figure 1)) by transiently displacing the tightly bound non-histone protein complexes (Azvolinsky et al., 2006; Ivessa et al., 2003; Ivessa et al., 2002; Ivessa et al., 2000). Since replication forks paused/stalled with similarly reduced rates at the inactive RFB, the 5S rDNA gene, and the silent replication origin in rDNA of old WT and old mutant rrm3 (age: ~10 cell divisions) cells, the age-dependent decrease in replication pausing at the RPS in rDNA does not likely depend on Rrm3p (Figure 2A, rrm3; ERCs: Figure S1B).

Figure 2. The reduced replication pausing/stalling at natural replication pause sites (RPS) in ribosomal DNA during aging is independent of Rrm3p and Sir2p, occurs on both chromosomal rDNA and ERCs, and is partly recapitulated under caloric-restricted growth conditions.

Figure 2

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Panels A and B display analyses of ClaI-digested nuclear rDNA replication intermediates by DNA 2D gels. See Figure 1 for probe, locations of the natural RPS in rDNA and quantitation of the replication intermediates. Standard errors are displayed. A. Young (not expressing the MEP) and aged (expressing the MEP) cells of the indicated strains (WT, rrm3, fob1, sir2) were used. Age and ERCs enrichment (old vs young; Figure S1B): WT (~8 cell divisions; 8-fold enrichment); rrm3 (~10 cell divisions; 22-fold enrichment); fob1 (~12 cell divisions; 2.1-fold enrichment); sir2 (~10 cell divisions; 27-fold enrichment). B. Cells (rrm3) were grown in 2% or 0.05% glucose (CR). Initiation of rDNA replication is reduced in CR-cells (Kwan et al., 2013).

We used the fob1 mutant to prevent the age-dependent accumulation of ERCs to address whether replication pausing at the natural RPS in rDNA occurs preferentially on chromosomal DNA in old cells (ERCs: Figure S1B) (Defossez et al., 1999; Lindstrom et al., 2011; Sinclair and Guarente, 1997). Like in old WT cells replication did not pause/stall at these sites in old fob1 mutant cells suggesting that the loss in pausing/stalling at the natural RPS in rDNA occurs on both chromosomes and ERCs (Figure 2A, fob1).

Mutants lacking the protein deacetylase Sir2p accumulate faster ERCs compared to WT (Figure S1B) and age faster (Kaeberlein et al., 1999). Though we expected increased replication pausing at the RPS in rDNA of old sir2 mutant cells, we found no significant difference between WT and sir2 mutant cells in replication pausing at the rDNA-RPS in young and old (~10 cell divisions) cells suggesting that Sir2p does not affect the loss in pausing during aging (Figure 2A, sir2).

Growth condition of caloric restriction (CR; 0.05% glucose), which extends lifespan (Lin et al., 2000), resulted in a significant loss in pausing at the 5S rDNA gene; pausing at the other RPS in rDNA was not affected (Figure 2B).

Reduced replication pausing at natural non-rDNA replication pause sites in aging and caloric-restricted cells

We observed that replication pauses less at several natural non-rDNA RPS including the silent replication origins ARS313/314 and ARS608, and the tRNA genes tRNAA, tRNAY, and tRNAF in old cells lacking Rrm3p (Figure 3A) (Azvolinsky et al., 2009; Ivessa et al., 2003). Replication pausing at tRNA genes (tRNAA, MEP rrm3) was also reduced in CR-cells (0.05% glucose; Figure 3B). These results suggest that reduced replication pausing occur genome-wide at natural RPS in aging cells and is partially recapitulated in CR-cells.

Figure 3. Reduced replication pausing at natural non-rDNA replication pause sites in aging and caloric-restricted cells.

Figure 3

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A. Young and old mutant rrm3 cells (~10 cell divisions; expressing the MEP) were analyzed by DNA 2D gels. Investigated sites: the silent replication origins ARS313/314 (*,**), and ARS608 (*), and the tRNA genes (*) tRNAA, tRNAY and tRNAF. Two different auto-radiographic exposures are displayed for the tRNAA and tRNAY genes. The area of the replication pause for the tRNAF gene is magnified in the upper right corner. The white triangles (young cells, tRNAY and tRNAF) indicate putative recombination intermediates/converging forks, which are reduced in old cells. B. CR mutant rrm3 cells were analyzed by DNA 2D gels at the tRNAA locus (two different auto-radiographic exposures are shown). In panels A and B quantitation of the replication intermediates was carried out as described in Figure 1. Standard errors are displayed.

Members of the non-histone binding complexes which occupy the replication pause sites exhibit diffused cytosolic and/or nuclear rim localization in aging cells

Both 5S rDNA genes and tRNA genes are bound by the RNA polymerase III preinitiation complex TFIIIC, and both rDNA and non-rDNA silent replication origins including the silent mating type loci are bound by the origin recognition complex (Orc), which all cause replication pausing (Deshpande and Newlon, 1996; Ivessa et al., 2003; Wyrick et al., 2001). Preventing the binding of the tightly-bound TFIIIC leads to unimpeded fork movement at the mutated tRNA gene (Deshpande and Newlon, 1996; Ivessa et al., 2003). We addressed whether these complexes (Orc, TFIIIC) are absent from the investigated sites in aging cells, thus causing the observed reduction in replication pausing (Figures 1 to 3).

We observed that Orc1p-13Myc is mainly localized in the nucleus in young cells. It is expressed in old cells, but has preferential cytosolic/nuclear rim localization in 20–30% of old cells (~15 cell divisions; Figures 4A and S2). In young cells, an average of about 93% of Orc1p overlapped with the nucleus (DAPI), whereas in old cells only 15% of Orc1p overlapped with the nuclear DAPI.

Figure 4. Members of the non-histone protein complexes ORC and TFIIIC which bind to natural replication pause sites (silent replication origins, tRNA and 5S rDNA genes) display largely diffused cytosolic or nuclear rim localization in aged cells.

Figure 4

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A. Orc1p-13Myc is detected by Western blotting in young and old (~15 cell divisions) diploid WT cells. Controls: expression of Sir2p is largely reduced in old cells, whereas the vacuolar protein Vma2p is still expressed in old cells (Dang et al., 2009; Lindstrom et al., 2011). The Ponceau S-stained western blot demonstrates equal loading (below). Immunofluorescent staining of young and old WT cells (right side): red, Orc1p-13Myc; blue, DNA (DAPI). Bar (bottom right): 5 μm. See also Figure S2. B. Strains expressing the nuclear transcription factors Tfc1-GFP and Tfc4-GFP in young and old (~8 cell divisions) haploid WT cells. Green: GFP; blue: calcofluor (age determination); grey: bright-field. Avidin-coated magnetic beads that bind to the biotin-labeled old cells can be seen as small, grainy dots surrounding the old cells. Bar: 5 μm. See also Figure S3. C. Chromatin-immuno-precipitation (ChIP) to asses binding of Tfc1p to 5S rDNA and tRNAA genes, of Fob1p to the rDNA-RFB and of Orc2p to the rDNA-ARS. A strain expressing Tfc1-GFP was used. Input DNA (before IP) was diluted 1:500, 1:1000, 1:2000, and 1:4000 prior to the PCR to demonstrate the linear range of the amplification. The samples were: young (log-phase cells, 2% glucose), old (8–10 cell divisions), CR (0.05% glucose), no crosslinking agent (i.e. formaldehyde, NO X) added to log-phase cells (2% glucose). Western blots of IP samples: Tfc1p-GFP (100 kDa), Orc2p (71 kDa), Fob1p (65 kDa). Amount of the PCR products of the input and IP samples were determined by an imaging system and ratios of IP/input were calculated. The IP/input value of the non-crosslinked control (NO X) was subtracted from each IP/input value (young, old, CR) and percentages were calculated for each indicated DNA location as followed: [([young, old or CR] - [NO X]) / ([young] - [NO X])] * 100. Standard errors are displayed. The raw IP/input data are shown in Figure S5. D. In our model we propose that in young cells non-histone protein complexes are successfully imported into the nucleus, bind to natural replication pause sites (RPS) and slow/stall DNA replication which may lead to DNA breakage. In contrast, in old cells the protein complexes are imported with a reduced rate (or not at all) into the nucleus and therefore do not bind to the RPS to impede DNA replication. Thus, the RPS may experience only reduced replication stress in aging cells.

To localize TFIIIC, we used strains expressing Tfc1p-GFP or Tfc4p-GFP (Breker et al., 2014). On average, about 90% of both Tfc1p and Tfc4p localized to the nucleus in young cells (Figure S3A). Aged cells (~8 cell divisions) displayed a heterogeneous expression and localization pattern (Figures 4B, S3B and C). Some cells had Tfc1p and Tfc4p highly expressed in the cytosol whereas other aged cells exhibit low diffused expression. In some aged cells undergoing mitosis, Tfc1p or Tfc4p was clearly localized to the nucleus in the daughter cell, but had diffused localization in the aged mother cell (Figures S3B (Tfc1p) and 4B (Tfc4p)). CR-cells (0.05% glucose) compared to cells grown in 2% glucose also had increased cytosolic localization of Tfc1p (Figure S4).

Chromatin-immuno-precipitation (ChIP) confirmed that in aged and CR-cells 5S rDNA and tRNA genes are less occupied by Tfc1p (Figures 4C and S5). The silent replication origin in rDNA was only less occupied by Orc2p (part of Orc) in aged cells, but not in CR-cells (Figures 4C and S5). The ChIP results are consistent with the above-shown 2D gel data.

Discussion

We demonstrate that in replicative aging yeast cells DNA replication is less pausing/stalling at natural difficult-to-replicate sites (i.e., replication pause sites, RPS). These results were unanticipated, since various forms of DNA damage (e.g., chromosome translocations) are increasing during aging. Since many of these genetic changes require DNA breakage, we expected an increase in pausing/stalling of replication, particularly at the RPS to induce breakage (Branzei and Foiani, 2010; Lindstrom et al., 2011; Vijg and Suh, 2013).

Tyler and colleagues determined that both inter- and intra-chromosomal translocations occur at high rates on the rDNA locus but also on the mitochondrial DNA locus during yeast replicative aging (Hu et al., 2014). Some of these translocations occur in the vicinity of replication origins (silent and active), and LTR and tRNA genes. If replication migrates unimpeded at these sites in old cells, then it will be interesting to investigate how breakage and translocation events are induced there. Since the general loss of histones induces transcription of almost all yeast genes, superhelical tension may increase and lead to chromosome breakage (Hu et al., 2014).

We also observed a reduction in replication pausing at the 5S rDNA gene and at a tRNA gene in CR-cells (Figures 2B and 3B). Reduced mRNA translation is one of several phenotypes of CR-cells (Wasko and Kaeberlein, 2014). Reductions in replication pausing at 5S rDNA genes and tRNA genes may also reflect that. Less replication pausing at chromosomal RPS and possibly less DNA breakage at those sites may contribute to the lifespan-extension observed in CR-cells, which, however, has not been fully addressed yet.

The examined RPS are occupied by non-histone protein complexes which cause replication pausing (Deshpande and Newlon, 1996; Ivessa et al., 2003). We demonstrate that members of these complexes (Orc1, Tfc1, Tfc4) have a cytosolic/nuclear rim distribution in aged cells, which is consistent with the observation that the nuclear protein import is impaired in aged cells (Lord et al., 2015). Similarly some proteins (e.g., the nuclear tRNA exporter Los1p) are also excluded from the nucleus in CR-cells (McCormick et al., 2015). We also demonstrate by ChIP that Orc and TFIIIC exhibit a reduced presence on the investigated RPS in aged and CR-cells (Figure 4C). This reduced presence of the non-histone protein complexes at the chromosomal RPS (rDNA, tRNA genes) may reduce replication pausing and potential DNA breakage, which may promote longevity (Figure 4D). However, to maintain essential cellular processes and to propagate, the nuclear protein import can only be reduced to a certain level.

Since the nuclear protein import becomes less efficient during aging, members of various non-histone protein complexes might be present in non-stoichiometric amounts in the nucleus leading to a failure in the proper formation of mature complexes that can bind DNA (Janssens et al., 2015). In contrast, Fob1p binds to the RFB in rDNA as a single protein thereby causing replication stalling. Therefore, we still observe replication stalling at the active RFB in rDNA of old cells although the stalling-rate is reduced compared to young cells (Figures 1 and 2). Although the nuclear import efficiency might vary for different nuclear proteins in different cells, the outcome would always be the same, i.e., a failure in the proper assembly of nuclear protein complexes such as Orc, since in each case different single subunits may be present in low amounts. Future studies will investigate whether the reduced replication pausing at the chromosomal RPS influences the occurrence of DNA breakage at non-fragile sites and whether the reduction in pausing (or stalling) indeed has an impact on longevity.

Experimental Procedures

Yeast strains and yeast methods

A list of used yeast strains and their construction is provided in the Supplemental Experimental Procedures.

Determination of the amount of ERCs and analysis of DNA replication intermediates

DNA was prepared by CsCl-gradient centrifugation (Ivessa, 2013). The amount of extrachromosomal rDNA circles (ERCs) and the structure of DNA replication intermediates were determined by 2D gels (Ivessa, 2013; Ivessa et al., 2003; Ivessa et al., 2000). Primer-sequences for the PCR amplifications of DNA probes are listed under Supplemental Information. The amounts of specific replication intermediates were determined by phosphor-imager analysis.

Other procedures

Conditions for the MEP, immunofluorescent staining, and fluorescence microscopy of yeast cells, FACS, and ChIP are outlined in the Supplemental Experimental Procedures.

Supplementary Material

1
2

Highlights and eTOC Blurb.

  • Reduced replication pausing at fragile sites in aging and caloric-restricted cells

  • Age-dependent reduction in pausing is independent of Rrm3 and Sir2

  • Fragile sites bound proteins are cytosolic in aged and caloric-restricted cells

  • Findings may have implications to understand chromosome rearrangements during aging

Acknowledgments

We are grateful to Dr. Daniel Gottschling (FHCRC, Seattle) for providing us with the MEP-expressing strains. We thank Christopher Brady for critically reading the paper. This work was supported by NIH grants R21AG047423 (to A.S.I.) and NIH SIG grant 1S0OD018103-01 (Rutgers NJMS Flow Cytometry and Immunology Core Laboratory) (to S.S.).

Footnotes

Supplemental information

This section includes Supplemental Results (five figures and figure legends), Supplemental Experimental Procedures, tables for primer sequences and used yeast strains.

Author Contribution

M.C., X.C., S.S, and A.S.I. conducted the experiments; S.S. carried out the FACS experiments; A.S.I. conceived and supervised the project, designed the experiments, analyzed the data, and wrote the paper.

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Supplementary Materials

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NIHMS828557-supplement-1.pdf (3.6MB, pdf)
2
NIHMS828557-supplement-2.pdf (32.8KB, pdf)

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