The increase in cell death rates in caloric restricted cells of the yeast helicase mutant rrm3 is Sir complex dependent.

Calorie restriction (CR), which is a reduction in calorie intake without malnutrition, usually extends lifespan and improves tissue integrity. This report focuses on the relationship between nuclear genomic instability and dietary-restriction and its effect on cell survival. We demonstrate that the cell survival rates of the genomic instability yeast mutant rrm3 change under metabolic restricted conditions. Rrm3 is a DNA helicase, chromosomal replication slows (and potentially stalls) in its absence with increased rates at over 1400 natural pause sites including sites within ribosomal DNA and tRNA genes. Whereas rrm3 mutant cells have lower cell death rates compared to wild type (WT) in growth medium containing normal glucose levels (i.e., 2%), under CR growth conditions cell death rates increase in the rrm3 mutant to levels, which are higher than WT. The silent-information-regulatory (Sir) protein complex and mitochondrial oxidative stress are required for the increase in cell death rates in the rrm3 mutant when cells are transferred from growth medium containing 2% glucose to CR-medium. The Rad53 checkpoint protein is highly phosphorylated in the rrm3 mutant in response to genomic instability in growth medium containing 2% glucose. Under CR, Rad53 phosphorylation is largely reduced in the rrm3 mutant in a Sir-complex dependent manner. Since CR is an adjuvant treatment during chemotherapy, which may target genomic instability in cancer cells, our studies may gain further insight into how these therapy strategies can be improved.

Aging: Fewer Obstacles to DNA Replication?

Many cancers are initiated by loss-of-heterozygosity (LOH) events that lead to the replacement of single, functional tumor suppressor genes by the mutant alleles. The underlying mechanisms, of why LOH rates increase with age, are not well understood. We discuss the possible involvement of difficult-to-replicate (fragile) chromosomal sites in this process.

Absence of Non-histone Protein Complexes at Natural Chromosomal Pause Sites Results in Reduced Replication Pausing in Aging Yeast Cells.

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 (RPSs) 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 RPSs, and is independent of the deacetylase Sir2p. Conditions of caloric restriction (CR), which extend life span, also cause reduced replication pausing at the 5S rDNA and at tRNA genes. In aged and CR cells, the RPSs 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.

Reversible mitochondrial DNA accumulation in nuclei of pluripotent stem cells.

According to the endosymbiotic hypothesis, the precursor of mitochondria invaded the precursor of eukaryotic cells, a process that began roughly 2 billion years ago. Since then, the majority of the genetic material translocated from the mitochondria to the nucleus, where now almost all mitochondrial proteins are expressed. Only a tiny amount of DNA remained in the mitochondria, known as mitochondrial DNA (mtDNA). In this study, we report that the transfer of mtDNA fragments to the nucleus of pluripotent stem cells is still ongoing. We show by in situ hybridization and agarose DNA two-dimensional gel technique that induced pluripotent stem (iPS) cells contain high levels of mtDNA in the nucleus. We found that a large proportion of the accumulated mtDNA sequences appear to be extrachromosomal. Accumulation of mtDNA in the nucleus is present not only in the iPS cells, but also in embryonic stem (ES) cells. However upon differentiation, the level of mtDNA in the nuclei of iPS and ES cells is substantially reduced. This reversible accumulation of mtDNA in the nucleus supports the notion that the nuclear copy number of mtDNA sequences may provide a novel mechanism by which chromosomal DNA is dynamically regulated in pluripotent stem cells.

Analysis of DNA structures from eukaryotic cells by two-dimensional native-native DNA agarose gel electrophoresis.

The neutral-neutral two-dimensional agarose gel technique is mainly used to determine the chromosomal positions where DNA replication starts, but it is also applied to visualize replication fork progression and breakage as well as intermediates in DNA recombination. Here we provide a step-by-step protocol to analyze the fairly underrepresented and fragile replication intermediates in yeast chromosomal DNA. The technique can also be adapted to analyze replication intermediates in chromosomal DNA of higher eukaryotic organisms.

Directionality of replication fork movement determined by two-dimensional native-native DNA agarose gel electrophoresis.

The analysis of replication intermediates by the neutral-neutral two-dimensional agarose gel technique allows determining the chromosomal positions where DNA replication initiates, whether replication forks pause or stall at specific sites, or whether two DNA molecules undergo DNA recombination events. This technique does not, however, immediately tell in which direction replication forks migrate through the DNA region under investigation. Here, we describe the procedure to determine the direction of replication fork progression by carrying out a restriction enzyme digest of DNA imbedded in agarose after the completion of the first dimension of a 2D gel.

Accumulation of linear mitochondrial DNA fragments in the nucleus shortens the chronological life span of yeast.

Translocation of mitochondrial DNA (mtDNA) fragments to the nucleus and insertion of those fragments into nuclear DNA has been observed in several organisms ranging from yeast to plants and mammals. Disruption of specific nuclear genes by de novo insertions of mtDNA fragments has even been linked to the initiation of several human diseases. Recently, we demonstrated that baker's yeast strains with high rates of mtDNA fragments migrating to the nucleus (yme1-1 mutant) exhibit short chronological life spans (CLS). The yeast CLS is determined by the survival of non-dividing cell populations. Here, we show that lack of the non-homologous-end-joining enzyme DNA ligase IV (DNL4) can rescue the short CLS of the yme1-1 mutant. In fission yeast, DNA ligase IV has been shown to be required for the capture of mtDNA fragments during the repair of double-stranded DNA breaks in nuclear DNA. In further analyses using pulse field gel and 2D gel electrophoresis we demonstrate that linear mtDNA fragments with likely nuclear localization accumulate in the yme1-1 mutant. The accumulation of the linear mtDNA fragments in the yme1-1 mutant is suppressed when Dnl4 is absent. We propose that the linear nuclear mtDNA fragments accelerate the aging process in the yme1-1 mutant cells by possibly affecting nuclear processes including DNA replication, recombination, and repair as well as transcription of nuclear genes. We speculate further that Dnl4 protein has besides its function as a ligase also a role in DNA protection. Dnl4 protein may stabilize the linear mtDNA fragments in the nucleus by binding to their physical ends. In the absence of Dnl4 protein the linear fragments are therefore unprotected and possibly degraded by nuclear nucleases.

Association of the yeast DNA helicase Pif1p with mitochondrial membranes and mitochondrial DNA.

Previously we demonstrated that the mitochondrial form of the yeast Pif1p DNA helicase, which we found to be attached to mitochondrial DNA (mtDNA), is required for the maintenance of mtDNA under genotoxic stress conditions. Here, we demonstrated that mitochondrial Pif1p is exclusively bound to mitochondrial membranes and part of an about 900kDa protein complex. Pif1p might be incorporated into this complex immediately after its translocation from the cytoplasm into the mitochondrial matrix. Pif1p as well as the mitochondrial DNA polymerase Mip1p could not be released from the mitochondrial membranes by digesting mtDNA with restriction enzymes in permeabilized mitochondria. In contrast, restriction enzyme-digested mtDNA fragments that were covered by the histone-like protein Abf2p were efficiently released from the permeabilized mitochondria. We propose that Pif1p as well as Mip1p are not only bound to mtDNA but also to the inner mitochondrial membrane either directly or indirectly via a protein complex. We also found that in the absence of mtDNA the total amount of cellular Pif1p is highly reduced.

The migration of mitochondrial DNA fragments to the nucleus affects the chronological aging process of Saccharomyces cerevisiae.

Migration of fragmented mitochondrial DNA (mtDNA) to the nucleus has been shown to occur in multiple species including yeast, plants, and mammals. Several human diseases, including Pallister-Hall syndrome and mucolipidosis, can be initiated by mtDNA insertion mutagenesis of nuclear DNA. In yeast, we demonstrated that the rate of mtDNA fragments translocating to the nucleus increases during chronological aging. The yeast chronological lifespan (CLS) is determined by the survival of nondividing cell populations. Whereas yeast strains with elevated migration rates of mtDNA fragments to the nucleus showed accelerated chronological aging, strains with decreased mtDNA transfer rates to the nucleus exhibited an extended CLS. Although one of the most popular theories of aging is the free radical theory, migration of mtDNA fragments to the nucleus may also contribute to the chronological aging process by possibly increasing nuclear genomic instability in cells with advanced age.

Loss of mitochondrial DNA under genotoxic stress conditions in the absence of the yeast DNA helicase Pif1p occurs independently of the DNA helicase Rrm3p.

How the cellular amount of mitochondrial DNA (mtDNA) is regulated under normal conditions and in the presence of genotoxic stress is less understood. We demonstrate that the inefficient mtDNA replication process of mutant yeast cells lacking the PIF1 DNA helicase is partly rescued in the absence of the DNA helicase RRM3. The rescue effect is likely due to the increase in the deoxynucleoside triphosphates (dNTPs) pool caused by the lack of RRM3. In contrast, the Pif1p-dependent mtDNA breakage in the presence and absence of genotoxic stress is not suppressed if RRM3 is lacking suggesting that this phenotype is likely independent of the dNTP pool. Pif1 protein (Pif1p) was found to stimulate the incorporation of dNTPs into newly synthesised mtDNA of gradient-purified mitochondria. We propose that Pif1p that acts likely as a DNA helicase in mitochondria affects mtDNA replication directly. Possible roles of Pif1p include the resolution of secondary DNA and/or DNA/RNA structures, the temporarily displacement of tightly bound mtDNA-binding proteins, or the stabilization of the mitochondrial replication complex during mtDNA replication.

The role of Pif1p, a DNA helicase in Saccharomyces cerevisiae, in maintaining mitochondrial DNA.

Mitochondrial DNA (mtDNA) is highly susceptible to oxidative and chemically induced damage, and these insults lead to a number of diseases. In Saccharomyces cerevisiae, the DNA helicase Pif1p is localized to the nucleus and mitochondria. We show that pif1 mutant cells are sensitive to ethidium bromide-induced damage and this mtDNA is prone to fragmentation. We also show that Pif1p associates with mtDNA. In pif1 mutant cells, mtDNA breaks at specific sites that exhibit Pif1-dependent recombination. We conclude that Pif1p participates in the protection from double-stranded (ds) DNA breaks or alternatively in the repair process of dsDNA breaks in mtDNA.

The Saccharomyces cerevisiae helicase Rrm3p facilitates replication past nonhistone protein-DNA complexes.

The Saccharomyces cerevisiae RRM3 gene encodes a 5' to 3' DNA helicase. While replication of most of the yeast genome was not dependent upon Rrm3p, in its absence, replication forks paused and often broke at an estimated 1400 discrete sites, including tRNA genes, centromeres, inactive replication origins, and transcriptional silencers. These replication defects were associated with activation of the intra-S phase checkpoint. Activation of the checkpoint was critical for viability of rrm3Delta cells, especially at low temperatures. Each site whose replication was affected by Rrm3p is assembled into a nonnucleosomal protein-DNA complex. At tRNA genes and the silent mating type loci, disruption of these complexes eliminated dependence upon Rrm3p. These data indicate that the Rrm3p DNA helicase helps replication forks traverse protein-DNA complexes, naturally occurring impediments that are encountered in each S phase.

Saccharomyces Rrm3p, a 5' to 3' DNA helicase that promotes replication fork progression through telomeric and subtelomeric DNA.

In wild-type Saccharomyces cerevisiae, replication forks slowed during their passage through telomeric C(1-3)A/TG(1-3) tracts. This slowing was greatly exacerbated in the absence of RRM3, shown here to encode a 5' to 3' DNA helicase. Rrm3p-dependent fork progression was seen at a modified Chromosome VII-L telomere, at the natural X-bearing Chromosome III-L telomere, and at Y'-bearing telomeres. Loss of Rrm3p also resulted in replication fork pausing at specific sites in subtelomeric DNA, such as at inactive replication origins, and at internal tracts of C(1-3)A/TG(1-3) DNA. The ATPase/helicase activity of Rrm3p was required for its role in telomeric and subtelomeric DNA replication. Because Rrm3p was telomere-associated in vivo, it likely has a direct role in telomere replication.