The Role of Replication Bypass Pathways in Dicentric Chromosome Formation in Budding Yeast

Gross chromosomal rearrangements (GCRs) are large scale changes to chromosome structure and can lead to human disease. We previously showed in Saccharomyces cerevisiae that nearby inverted repeat sequences (∼20–200 bp of homology, separated by ∼1–5 kb) frequently fuse to form unstable dicentric and acentric chromosomes. Here we analyzed inverted repeat fusion in mutants of three sets of genes. First, we show that genes in the error-free postreplication repair (PRR) pathway prevent fusion of inverted repeats, while genes in the translesion branch have no detectable role. Second, we found that siz1 mutants, which are defective for Srs2 recruitment to replication forks, and srs2 mutants had opposite effects on instability. This may reflect separate roles for Srs2 in different phases of the cell cycle. Third, we provide evidence for a faulty template switch model by studying mutants of DNA polymerases; defects in DNA pol delta (lagging strand polymerase) and Mgs1 (a pol delta interacting protein) lead to a defect in fusion events as well as allelic recombination. Pol delta and Mgs1 may collaborate either in strand annealing and/or DNA replication involved in fusion and allelic recombination events. Fourth, by studying genes implicated in suppression of GCRs in other studies, we found that inverted repeat fusion has a profile of genetic regulation distinct from these other major forms of GCR formation.ALL organisms are prone to large-scale changes (gross chromosomal rearrangements, GCRs) to their genomes that include deletions, inversions, and translocations. These large-scale changes are thought to drive evolutionary events, such as speciation, and contribute to human pathology such as Pelziaeus-Merzbacher syndrome and other genetic disorders (Lee et al. 2007; Stankiewicz and Lupski 2010). Thus, a firm understanding of how cells normally prevent such rearrangements, and how they accumulate, is critical to our understanding of both evolution and pathology.GCRs arise by many different mechanisms, and there is growing evidence that errors during DNA replication are a major source (Myung et al. 2001; Admire et al. 2006; Mizuno et al. 2009). Errors are thought to arise when replication forks encounter “lesions” on the template strand. Lesions can consist of protein complexes bound to DNA or lesions in the DNA itself. Replication forks bypass lesions by several different mechanisms that are still poorly understood (Atkinson and McGlynn 2009; Weinert et al. 2009). We believe that understanding lesion bypass mechanisms is central to understanding both how GCRs are prevented and how they form when lesion bypass mechanisms fail.All lesion bypass pathways utilize sequence homology to restart replication (Atkinson and McGlynn 2009; Weinert et al. 2009). Use of sequence homology during restart may limit the frequency of GCRs, as it lowers the probability of annealing to nonallelic sequences. Repetitive sequences present a problem because lesion bypass at sites near repetitive sequences may lead to annealing of nonallelic sequences and thus to GCR formation (Lemoine et al. 2005; Narayanan et al. 2006; Argueso et al. 2008). Indeed in yeast and in other organisms, GCRs occur frequently in repeat sequences (Dunham et al. 2002; Argueso et al. 2008; Di Rienzi et al. 2009). Some rearrangements do occur between so-called “single-copy sequences” with either no homology or limited homology (microhomologies of 5–9 bp; Myung et al. 2001; Kolodner et al. 2002; Putnam et al. 2005) though evidence suggests these rearrangements occur less frequently than rearrangements between repetitive sequences (Putnam et al. 2009). Interestingly, it has been shown that some genes are required to prevent the fusion of repetitive elements yet have no effect on rearrangements between single-copy sequences (Putnam et al. 2009). Currently it is not clear how these pathways act to suppress repeat-mediated events and why they are not required to prevent rearrangements between single-copy sequences.Our current understanding of the mechanisms underlying GCR formation is mostly derived from assays designed to detect specific changes to yeast chromosomes (Chen and Kolodner 1999; Myung et al. 2001; Huang and Koshland 2003; Lambert et al. 2005; Rattray et al. 2005; Admire et al. 2006; Narayanan et al. 2006; Schmidt et al. 2006; Smith et al. 2007; Pannunzio et al. 2008; Payen et al. 2008; Paek et al. 2009; Mizuno et al. 2009). Previously we reported on GCR formation in the budding yeast Saccharomyces cerevisiae using an assay we developed. We found that a major source of genome instability involves the fusion of nearby inverted repeats (with ∼20–200 bp of sequence homology, separated by 1–5 kb) to form either dicentric or acentric chromosomes (Figure 1D; Paek et al. 2009). We also found that fusion of inverted repeats is general: fusion occurred between inverted repeats at all five different locations tested on four different yeast chromosomes, as well as between synthetic inverted repeats (Paek et al. 2009). Genetic data suggest that these events most likely occur during replication of DNA (Admire et al. 2006). Further genetic analysis suggested that the mechanism of inverted repeat fusion differed from that of direct repeat recombination, in that inverted repeat fusion did not require genes involved in homologous recombination (HR) or single-strand annealing (SSA) pathways (Paek et al. 2009). In addition, fusion events are unlikely to involve double-strand breaks (DSBs), as genes in the nonhomologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ) are not required for fusion events (Paek et al. 2009). Indeed gene knockouts in the HR (RAD52, RAD51, and RAD59), SSA (RAD52 and RAD1) and postreplication repair (PRR) (RAD18) pathways actually increased the frequency of fusion of an inverted repeat on chromosome (Chr) VII (Paek et al. 2009); these pathways normally suppress inverted repeat fusion.Open in a separate windowFigure 1.—Experimental setup for the detection of inverted repeat fusion and chromosome instability. Objects are not drawn to scale. (A) The starting strain has two copies of Chr VII. One copy contains the CAN1 gene, ADE6, ade3, while the other copy is ade6, ADE3. Cells are plated to canavanine, and three types of colonies are formed: (B) Allelic recombinants are round in appearance and are Ade⁺; (C) colonies that form by loss of Chr VII are round in appearance and Ade⁻; and (D) cells that contain unstable dicentric chromosomes form by the fusion of inverted repeats. One specific case of this fusion (the S2/S3 dicentric) is shown within braces. Cells with dicentrics form mixed colonies, which contain allelic recombinants, chromosome loss events, as well as a translocation between D7 and D11. The bar in the S2/S3 repeat represents a fusion junction. (E) The specific dicentric is detected by dicentric primers DP1 and DP2 and (F) a monocentric translocation that is detected with translocation primers TP1 and TP2.To further our previous studies, we analyzed three groups of genes implicated in the maintenance of genome stability. We tested how these genes affect the overall stability of Chr VII, focusing on the fusion of nearby inverted repeats to form a specific dicentric Chr VII and the resolution of the dicentric into a monocentric translocation (which we term the 403–535 translocation; Figure 1, D–F). First, we analyzed several genes in the PRR pathway and found that error-free bypass, but not translesion synthesis, is required for the prevention of inverted repeat fusion. Surprisingly, we found that siz1 mutants, which are defective for Srs2 recruitment to replication forks, and srs2 mutants had opposite effects on instability. This may reflect separate roles for Srs2 in different phases of the cell cycle. Second, we analyzed several mutations in genes that are associated with replication forks. We found that mutants in POL3 (polymerase delta) and MGS1 (encoding a single-strand annealing protein, which binds polymerase delta) significantly reduced the frequency of dicentric formation and allelic recombinants that arise in the checkpoint mutant rad9 (Giot et al. 1997; Hishida et al. 2001; Paek et al. 2009). Finally we studied genes associated with rearrangements involving repeats or single-copy sequences, as well as a subset of mutants involved in recombination. Generally, we find that the mechanisms of nearby inverted repeat fusion are distinct from mechanisms fusing longer repeats or single-copy sequences.     

Temperature-Dependent Modulation of Chromosome Segregation in msh4 Mutants of Budding Yeast

Background

In many organisms, homologous chromosomes rely upon recombination-mediated linkages, termed crossovers, to promote their accurate segregation at meiosis I. In budding yeast, the evolutionarily conserved mismatch-repair paralogues, Msh4 and Msh5, promote crossover formation in conjunction with several other proteins, collectively termed the Synapsis Initiation Complex (SIC) proteins or ‘ZMM’s (Zip1-Zip2-Zip3-Zip4-Spo16, Msh4-Msh5, Mer3). zmm mutants show decreased levels of crossovers and increased chromosome missegregation, which is thought to cause decreased spore viability.

Principal Findings

In contrast to other ZMM mutants, msh4 and msh5 mutants show improved spore viability and chromosome segregation in response to elevated temperature (23°C versus 33°C). Crossover frequencies in the population of viable spores in msh4 and msh5 mutants are similar at both temperatures, suggesting that temperature-mediated chromosome segregation does not occur by increasing crossover frequencies. Furthermore, meiotic progression defects at elevated temperature do not select for a subpopulation of cells with improved segregation. Instead, another ZMM protein, Zip1, is important for the temperature-dependent improvement in spore viability.

Conclusions

Our data demonstrate interactions between genetic (zmm status) and environmental factors in determining chromosome segregation.     

Chromosome Segregation in Budding Yeast: Sister Chromatid Cohesion and Related Mechanisms

Studies on budding yeast have exposed the highly conserved mechanisms by which duplicated chromosomes are evenly distributed to daughter cells at the metaphase–anaphase transition. The establishment of proteinaceous bridges between sister chromatids, a function provided by a ring-shaped complex known as cohesin, is central to accurate segregation. It is the destruction of this cohesin that triggers the segregation of chromosomes following their proper attachment to microtubules. Since it is irreversible, this process must be tightly controlled and driven to completion. Furthermore, during meiosis, modifications must be put in place to allow the segregation of maternal and paternal chromosomes in the first division for gamete formation. Here, I review the pioneering work from budding yeast that has led to a molecular understanding of the establishment and destruction of cohesion.     

Budding Yeast DNA Damage Adaptation Mutants Exhibit Defects in Mitotic Exit

In the presence of double strand breaks, DNA damage checkpoint halts cell cycle progression. However, cells ultimately escape the checkpoint arrest and re-enter cell cycle in the presence of irreparable DNA damage. cdc5-ad was identified as a mutant that fails to adapt to the cell cycle arrest induced by DNA damage checkpoint. In budding yeast, Cdc5 protein kinase is a component of both MEN and FEAR pathways that are required for mitotic exit. It remains unclear whether the adaptation defect of cdc5-ad mutant cells is related to the function of Cdc5 in mitotic exit. Here we present evidence indicating that cdc5-ad mutant cells exhibit defects in mitotic exit. cdc5-ad mutant cells are sensitive to high dosage of Amn1, a negative regulator of MEN. It also shows synthetic growth defects with mutants in MEN pathway. Moreover, mutants in FEAR pathway exhibit defects in DNA damage adaptation. Thus, we conclude that the compromised mitotic exit pathway contributes to DNA damage adaptation defects in cdc5-ad mutant cells.     

Kinetochore Function and Chromosome Segregation Rely on Critical Residues in Histones H3 and H4 in Budding Yeast

Accurate chromosome segregation requires that sister kinetochores biorient and attach to microtubules from opposite poles. Kinetochore biorientation relies on the underlying centromeric chromatin, which provides a platform to assemble the kinetochore and to recruit the regulatory factors that ensure the high fidelity of this process. To identify the centromeric chromatin determinants that contribute to chromosome segregation, we performed two complementary unbiased genetic screens using a library of budding yeast mutants in every residue of histone H3 and H4. In one screen, we identified mutants that lead to increased loss of a nonessential chromosome. In the second screen, we isolated mutants whose viability depends on a key regulator of biorientation, the Aurora B protein kinase. Nine mutants were common to both screens and exhibited kinetochore biorientation defects. Four of the mutants map near the unstructured nucleosome entry site, and their genetic interaction with reduced IPL1 can be suppressed by increasing the dosage of SGO1, a key regulator of biorientation. In addition, the composition of purified kinetochores was altered in six of the mutants. Together, this work identifies previously unknown histone residues involved in chromosome segregation and lays the foundation for future studies on the role of the underlying chromatin structure in chromosome segregation.     

Self-Regulating Model for Control of Replication Origin Firing in Budding Yeast

A major research area concentrates on understanding the regulation of replication origin firing. It is now appreciated that checkpoint signaling participates in this controlled process and that defects in such signaling systems affect genome integrity. Inhibition of replication origin firing is most obviously apparent under conditions of replication stress, but origin firing must also be regulated on a minute-by-minute basis as cells progress normally through an unabated S-phase. Here we summarize a straightforward model to account for how origin firing could be controlled by a self-regulating system.     

Evidence for Two Different Regulatory Mechanisms Linking Replication and Segregation of Vibrio cholerae Chromosome II

Understanding the mechanisms that coordinate replication initiation with subsequent segregation of chromosomes is an important biological problem. Here we report two replication-control mechanisms mediated by a chromosome segregation protein, ParB2, encoded by chromosome II of the model multichromosome bacterium, Vibrio cholerae. We find by the ChIP-chip assay that ParB2, a centromere binding protein, spreads beyond the centromere and covers a replication inhibitory site (a 39-mer). Unexpectedly, without nucleation at the centromere, ParB2 could also bind directly to a related 39-mer. The 39-mers are the strongest inhibitors of chromosome II replication and they mediate inhibition by binding the replication initiator protein. ParB2 thus appears to promote replication by out-competing initiator binding to the 39-mers using two mechanisms: spreading into one and direct binding to the other. We suggest that both these are novel mechanisms to coordinate replication initiation with segregation of chromosomes.     

Time to Be Versatile: Regulation of the Replication Timing Program in Budding Yeast

Eukaryotic replication origins are activated at different times during the S phase of the cell cycle, following a temporal program that is stably transmitted to daughter cells. Although the mechanisms that control initiation at the level of individual origins are now well understood, much less is known on how cells coordinate replication at hundreds of origins distributed on the chromosomes. In this review, we discuss recent advances shedding new light on how this complex process is regulated in the budding yeast Saccharomyces cerevisiae. The picture that emerges from these studies is that replication timing is regulated in cis by mechanisms modulating the chromatin structure and the subnuclear organization of origins. These mechanisms do not affect the licensing of replication origins but determine their ability to compete for limiting initiation factors, which are recycled from early to late origins throughout the length of the S phase.     

Direct and Indirect Control of the Initiation of Meiotic Recombination by DNA Damage Checkpoint Mechanisms in Budding Yeast

Meiotic recombination plays an essential role in the proper segregation of chromosomes at meiosis I in many sexually reproducing organisms. Meiotic recombination is initiated by the scheduled formation of genome-wide DNA double-strand breaks (DSBs). The timing of DSB formation is strictly controlled because unscheduled DSB formation is detrimental to genome integrity. Here, we investigated the role of DNA damage checkpoint mechanisms in the control of meiotic DSB formation using budding yeast. By using recombination defective mutants in which meiotic DSBs are not repaired, the effect of DNA damage checkpoint mutations on DSB formation was evaluated. The Tel1 (ATM) pathway mainly responds to unresected DSB ends, thus the sae2 mutant background in which DSB ends remain intact was employed. On the other hand, the Mec1 (ATR) pathway is primarily used when DSB ends are resected, thus the rad51 dmc1 double mutant background was employed in which highly resected DSBs accumulate. In order to separate the effect caused by unscheduled cell cycle progression, which is often associated with DNA damage checkpoint defects, we also employed the ndt80 mutation which permanently arrests the meiotic cell cycle at prophase I. In the absence of Tel1, DSB formation was reduced in larger chromosomes (IV, VII, II and XI) whereas no significant reduction was found in smaller chromosomes (III and VI). On the other hand, the absence of Rad17 (a critical component of the ATR pathway) lead to an increase in DSB formation (chromosomes VII and II were tested). We propose that, within prophase I, the Tel1 pathway facilitates DSB formation, especially in bigger chromosomes, while the Mec1 pathway negatively regulates DSB formation. We also identified prophase I exit, which is under the control of the DNA damage checkpoint machinery, to be a critical event associated with down-regulating meiotic DSB formation.     

Shared Genetic Pathways Contribute to the Tolerance of Endogenous and Low-Dose Exogenous DNA Damage in Yeast

DNA damage that escapes repair and blocks replicative DNA polymerases is tolerated by bypass mechanisms that fall into two general categories: error-free template switching and error-prone translesion synthesis. Prior studies of DNA damage responses in Saccharomyces cerevisiae have demonstrated that repair mechanisms are critical for survival when a single, high dose of DNA damage is delivered, while bypass/tolerance mechanisms are more important for survival when the damage level is low and continuous (acute and chronic damage, respectively). In the current study, epistatic interactions between DNA-damage tolerance genes were examined and compared when haploid yeast cells were exposed to either chronic ultraviolet light or chronic methyl methanesulfonate. Results demonstrate that genes assigned to error-free and error-prone bypass pathways similarly promote survival in the presence of each type of chronic damage. In addition to using defined sources of chronic damage, rates of spontaneous mutations generated by the Pol ζ translesion synthesis DNA polymerase (complex insertions in a frameshift-reversion assay) were used to infer epistatic interactions between the same genes. Similar epistatic interactions were observed in analyses of spontaneous mutation rates, suggesting that chronic DNA-damage responses accurately reflect those used to tolerate spontaneous lesions. These results have important implications when considering what constitutes a safe and acceptable level of exogenous DNA damage.     

Identification and Cloning of the Chl4 Gene Controlling Chromosome Segregation in Yeast

A collection of chl mutants characterized by decreased fidelity of chromosome transmission and by minichromosome nondisjunction in mitosis was examined for the ability to maintain nonessential dicentric plasmids. In one of the seven mutants analyzed, chl4, dicentric plasmids did not depress cell division. Moreover, nonessential dicentric plasmids were maintained stably without any rearrangements during many generations in the chl4 mutant. The rate of mitotic heteroallelic recombination in the chl4 mutant was not increased compared to that in an isogenic wild-type strain. Analysis of the segregation of a marked chromosome indicated that sister chromatid nondisjunction and sister chromatid loss contributed equally to chromosome malsegregation in the chl4 mutant. A genomic clone of CHL4 was isolated by complementation of the chl4-1 mutation and was physically mapped to the right arm of chromosome IV near the SUP2 gene. Nucleotide sequence analysis of CHL4 clone revealed a 1.4-kb open reading frame coding for a 53-kD predicted protein which does not have homology to published proteins. A strain containing a null allele of CHL4 is viable under standard growth conditions but has a temperature-sensitive phenotype (conditional lethality at 36°). We suggest that the CHL4 gene is required for kinetochore function in the yeast Saccharomyces cerevisiae.     

Suppression of Chromosome Healing and Anticheckpoint Pathways in Yeast Postsenescence Survivors

Telomere repeat-like sequences at DNA double-strand breaks (DSBs) inhibit DNA damage signaling and serve as seeds to convert DSBs to new telomeres in mutagenic chromosome healing pathways. We find here that the response to seed-containing DSBs differs fundamentally between budding yeast (Saccharomyces cerevisiae) cells that maintain their telomeres via telomerase and so-called postsenescence survivors that use recombination-based alternative lengthening of telomere (ALT) mechanisms. Whereas telomere seeds are efficiently elongated by telomerase, they remain remarkably stable without de novo telomerization or extensive end resection in telomerase-deficient (est2Δ, tlc1Δ) postsenescence survivors. This telomere seed hyper-stability in ALT cells is associated with, but not caused by, prolonged DNA damage checkpoint activity (RAD9, RAD53) compared to telomerase-positive cells or presenescent telomerase-negative cells. The results indicate that both chromosome healing and anticheckpoint activity of telomere seeds are suppressed in yeast models of ALT pathways.     

Checkpoint-Dependent Regulation of Origin Firing and Replication Fork Movement in Response to DNA Damage in Fission Yeast

To elucidate the checkpoint mechanism responsible for slowing passage through S phase when fission yeast cells are treated with the DNA-damaging agent methyl methanesulfonate (MMS), we carried out two-dimensional gel analyses of replication intermediates in cells synchronized by cdc10 block (in G₁) followed by release into synchronous S phase. The results indicated that under these conditions early-firing centromeric origins were partially delayed but late-firing telomeric origins were not delayed. Replication intermediates persisted in MMS-treated cells, suggesting that replication fork movement was inhibited. These effects were dependent on the Cds1 checkpoint kinase and were abolished in cells overexpressing the Cdc25 phosphatase, suggesting a role for the Cdc2 cyclin-dependent kinase. We conclude that both partial inhibition of the firing of a subset of origins and inhibition of replication fork movement contribute to the slowing of S phase in MMS-treated fission yeast cells.In response to low levels of the DNA-alkylating agent methyl methanesulfonate (MMS), wild-type yeast cells slow their progression through S phase, while cells lacking the appropriate upstream checkpoint kinase (Mec1 in the budding yeast Saccharomyces cerevisiae; Rad3 in the fission yeast Schizosaccharomyces pombe) or the appropriate downstream checkpoint kinase (Rad53 in budding yeast, Cds1 in fission yeast) fail to do so. Other DNA-damaging agents also cause a checkpoint-dependent slowing of S phase, in vertebrates as well as in yeasts. This slowing of S phase in response to DNA damage is sometimes called the “intra-S-phase” checkpoint (3, 6, 22, 23, 26, 28, 36, 37, 45, 53). Here we shall refer to it as the “S-phase damage” checkpoint.Prior to this report, the downstream portions of the checkpoint pathway(s) that slow S phase in response to DNA damage in fission yeast were unclear. However, the upstream portions of these pathways in fission yeast and other organisms have been partially elucidated, and downstream mechanisms in other organisms have been partially clarified. In all studied systems, upon detection of DNA damage in S phase, checkpoint proteins initiate a phosphorylation cascade that ultimately leads to slowing of replication. Upstream signaling in these systems involves the activation of one or more of the phosphatidylinositol-3-kinase-like protein kinases (PIK kinases; ATR and/or ATM in humans, Mec1 and/or Tel1 in budding yeast, and Rad3 in fission yeast). The activated PIK kinases then phosphorylate several proteins, including certain Ser/Thr kinases (Chk1 and/or Chk2 in humans, Rad53 in budding yeast, and Cds1 in fission yeast). These kinases, in turn, phosphorylate other substrates that, directly or indirectly, mediate the slowing of S phase (reviewed in reference 3).In budding yeast, two different mechanisms were shown to slow S phase upon DNA damage by MMS. Of these, one mechanism, inhibition of late-firing origins, depended on the Mec1-Rad53 checkpoint pathway (45, 53), while the other mechanism, inhibition of replication forks, appeared to be a direct consequence of DNA damage rather than a result of checkpoint activation (53). Tercero and Diffley (53) found that, in MMS-treated cells with mutations in the RAD53 gene, unregulated origin firing compensated for checkpoint-independent replication fork slowing, thus permitting a relatively normal overall rate of DNA synthesis. The mechanism by which the Rad53 protein modulates late origin activity is not yet clear, but one possibility is inhibition (by Rad53-catalyzed phosphorylation) of Dbf4, the regulatory subunit of the Cdc7-Dbf4 kinase, which is essential for initiation of replication (7, 8, 14, 55).In vertebrates, at least three different pathways have been shown to contribute to the slowing of S phase after DNA damage. In some cases checkpoint-mediated phosphorylation of Dbf4 inhibits progression through S phase by downregulating origin firing (7, 14), as may take place in budding yeast. In other cases, checkpoint-mediated phosphorylation leads to inhibition and destruction of the protein phosphatase Cdc25A, which is an activator of Cdk2. Cdk2 is the S-phase-specific cyclin-dependent kinase. Cdk2 activity is crucial for initiation of DNA replication and is modulated by inhibitory phosphorylation at Tyr-15. Cdc25A activates Cdk2 by dephosphorylating Tyr-15. Thus, when Cdc25A is phosphorylated by checkpoint kinases after DNA damage and subsequently destroyed, Cdk2 can no longer promote initiation of DNA replication (9, 27). The third mechanism by which vertebrate cells can slow progression through S phase is inhibition of replication fork movement. In vertebrate cells, slowing of replication forks in response to DNA damage is frequently checkpoint dependent; in contrast, in budding yeast, such slowing appeared to be checkpoint independent. In the tested cases, fork slowing has proved to be dependent on the PIK kinase ATR (homologous to budding yeast Mec1 and fission yeast Rad3) and on the Ser/Thr kinase Chk1 (a functional analogue of budding yeast''s Rad53 and fission yeast''s Cds1). In each of these cases, the checkpoint response to DNA damage led to inhibition of origin firing as well as to inhibition of replication fork movement (42, 44, 54). The precise mechanism leading to slowing of replication fork movement has not been fully worked out, but the mechanism appears to involve interactions between Chk1 and the proteins Tim and Tipin (54), whose yeast homologues (Swi1 and Swi3 in fission yeast, Tof1 and Csm3 in budding yeast) form a “replication fork protection complex” that is associated with replication forks (19, 33).Although it is clear that slowing of S phase in response to MMS-induced DNA damage in fission yeast requires both the Rad3 and Cds1 kinases, the pathways operating downstream of Cds1 have been uncertain. We obtained results indicating that Cdc25, which was already known to be a target of Cds1 in hydroxyurea (HU)-treated cells, is also a target of Cds1 in MMS-treated cells, because both overproduction of Cdc25 and conversion of Tyr-15 on Cdc2 (the major cyclin-dependent kinase of fission yeast; also known as Cdk1) to a nonphosphorylatable residue (Cdc2-Y15F; this mutation rendered Cdc2 constitutively active) were sufficient to prevent MMS-induced slowing of S phase (23). We concluded that, in fission yeast, the Rad3→Cds1⊣Cdc25→Cdc2 pathway forms a checkpoint signaling module very similar to the corresponding one of vertebrates. However, Kommajosyula and Rhind were not able to repeat our observations regarding the roles of Cdc25 and Cdc2 (22), so the relevance of Cdc25 and Cdc2 to checkpoint-induced slowing of S phase in fission yeast has remained uncertain until now. In addition, whether S phase in MMS-treated fission yeast cells is slowed by inhibition of origin firing, by reduction in rate of fork movement, or by a combination of these has been equally unclear.In order to resolve these issues, we initiated the series of experiments reported in this paper. To measure the rate of progression through S phase, we followed S phase by flow cytometry and by two-dimensional (2D) gel electrophoresis in cells released from a G₁ block (achieved by incubating cells bearing a cdc10 temperature-sensitive mutation at the restrictive temperature, then releasing to the permissive temperature [21, 23]). We found that, in MMS-treated, checkpoint-competent cells, the firing of early origins near centromeres was partially delayed but that the firing of late origins near telomeres was unaffected. Furthermore, the lifetimes of replication intermediates (RIs) were prolonged, consistent with slowing of replication forks. These effects were completely abrogated both in cells lacking the Cds1 kinase and in cells overproducing the Cdc25 phosphatase, showing that these effects were checkpoint dependent and that the relevant checkpoint pathway probably involved inhibition of Cdc25.     

Non-repair Pathways for Minimizing Protein Isoaspartyl Damage in the Yeast Saccharomyces cerevisiae

The spontaneous degradation of asparaginyl and aspartyl residues to isoaspartyl residues is a common type of protein damage in aging organisms. Although the protein-l-isoaspartyl (d-aspartyl) O-methyltransferase (EC 2.1.1.77) can initiate the repair of l-isoaspartyl residues to l-aspartyl residues in most organisms, no gene homolog or enzymatic activity is present in the budding yeast Saccharomyces cerevisiae. Therefore, we used biochemical approaches to elucidate how proteins containing isoaspartyl residues are metabolized in this organism. Surprisingly, the level of isoaspartyl residues in yeast proteins (50–300 pmol of isoaspartyl residues/mg of protein extract) is comparable with organisms with protein-l-isoaspartyl (d-aspartyl) O-methyltransferase, suggesting a novel regulatory pathway. Interfering with common protein quality control mechanisms by mutating and inhibiting the proteasomal and autophagic pathways in vivo did not increase isoaspartyl residue levels compared with wild type or uninhibited cells. However, the inhibition of metalloproteases in in vitro aging experiments by EDTA resulted in an ∼3-fold increase in the level of isoaspartyl-containing peptides. Characterization by mass spectrometry of these peptides identified several proteins involved in metabolism as targets of isoaspartyl damage. Further analysis of these peptides revealed that many have an N-terminal isoaspartyl site and originate from proteins with short half-lives. These results suggest that one or more metalloproteases participate in limiting isoaspartyl formation by robust proteolysis.     

Replication of an Autonomous Human Parvovirus in Non-dividing Human Airway Epithelium Is Facilitated through the DNA Damage and Repair Pathways

Human bocavirus 1 (HBoV1) belongs to the genus Bocaparvovirus of the Parvoviridae family, and is an emerging human pathogenic respiratory virus. In vitro, HBoV1 infects well-differentiated/polarized primary human airway epithelium (HAE) cultured at an air-liquid interface (HAE-ALI). Although it is well known that autonomous parvovirus replication depends on the S phase of the host cells, we demonstrate here that the HBoV1 genome amplifies efficiently in mitotically quiescent airway epithelial cells of HAE-ALI cultures. Analysis of HBoV1 DNA in infected HAE-ALI revealed that HBoV1 amplifies its ssDNA genome following a typical parvovirus rolling-hairpin DNA replication mechanism. Notably, HBoV1 infection of HAE-ALI initiates a DNA damage response (DDR) with activation of all three phosphatidylinositol 3-kinase–related kinases (PI3KKs). We found that the activation of the three PI3KKs is required for HBoV1 genome amplification; and, more importantly, we identified that two Y-family DNA polymerases, Pol η and Pol κ, are involved in HBoV1 genome amplification. Overall, we have provided an example of de novo DNA synthesis (genome amplification) of an autonomous parvovirus in non-dividing cells, which is dependent on the cellular DNA damage and repair pathways.     

Active Role of a Human Genomic Insert in Replication of a Yeast Artificial Chromosome

Yeast artificial chromosomes (YACs) are a common tool for cloning eukaryotic DNA. The manner by which large pieces of foreign DNA are assimilated by yeast cells into a functional chromosome is poorly understood, as is the reason why some of them are stably maintained and some are not. We examined the replication of a stable YAC containing a 240-kb insert of DNA from the human T-cell receptor beta locus. The human insert contains multiple sites that serve as origins of replication. The activity of these origins appears to require the yeast ARS consensus sequence and, as with yeast origins, additional flanking sequences. In addition, the origins in the human insert exhibit a spacing, a range of activation efficiencies, and a variation in times of activation during S phase similar to those found for normal yeast chromosomes. We propose that an appropriate combination of replication origin density, activation times, and initiation efficiencies is necessary for the successful maintenance of YAC inserts.