Comprehensive mutational analysis of the checkpoint signaling function of Rpa1/Ssb1 in fission yeast.

Replication protein A (RPA) is a heterotrimeric complex and the major single-strand DNA (ssDNA) binding protein in eukaryotes. It plays important roles in DNA replication, repair, recombination, telomere maintenance, and checkpoint signaling. Because RPA is essential for cell survival, understanding its checkpoint signaling function in cells has been challenging. Several RPA mutants have been reported previously in fission yeast. None of them, however, has a defined checkpoint defect. A separation-of-function mutant of RPA, if identified, would provide significant insights into the checkpoint initiation mechanisms. We have explored this possibility and carried out an extensive genetic screen for Rpa1/Ssb1, the large subunit of RPA in fission yeast, looking for mutants with defects in checkpoint signaling. This screen has identified twenty-five primary mutants that are sensitive to genotoxins. Among these mutants, two have been confirmed partially defective in checkpoint signaling primarily at the replication fork, not the DNA damage site. The remaining mutants are likely defective in other functions such as DNA repair or telomere maintenance. Our screened mutants, therefore, provide a valuable tool for future dissection of the multiple functions of RPA in fission yeast.

SUMO-targeted ubiquitin ligase activity can either suppress or promote genome instability, depending on the nature of the DNA lesion.

The posttranslational modifiers SUMO and ubiquitin critically regulate the DNA damage response (DDR). Important crosstalk between these modifiers at DNA lesions is mediated by the SUMO-targeted ubiquitin ligase (STUbL), which ubiquitinates SUMO chains to generate SUMO-ubiquitin hybrids. These SUMO-ubiquitin hybrids attract DDR proteins able to bind both modifiers, and/or are degraded at the proteasome. Despite these insights, specific roles for SUMO chains and STUbL in the DDR remain poorly defined. Notably, fission yeast defective in SUMO chain formation exhibit near wild-type resistance to genotoxins and moreover, have a greatly reduced dependency on STUbL activity for DNA repair. Based on these and other data, we propose that a critical role of STUbL is to antagonize DDR-inhibitory SUMO chain formation at DNA lesions. In this regard, we identify a SUMO-binding Swi2/Snf2 translocase called Rrp2 (ScUls1) as a mediator of the DDR defects in STUbL mutant cells. Therefore, in support of our proposal, SUMO chains attract activities that can antagonize STUbL and other DNA repair factors. Finally, we find that Taz1TRF1/TRF2-deficiency triggers extensive telomeric poly-SUMOylation. In this setting STUbL, together with its cofactor Cdc48p97, actually promotes genomic instability caused by the aberrant processing of taz1Δ telomeres by DNA repair factors. In summary, depending on the nature of the initiating DNA lesion, STUbL activity can either be beneficial or harmful.

RPA prevents G-rich structure formation at lagging-strand telomeres to allow maintenance of chromosome ends.

Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in DNA replication, recombination, and repair. In fission yeast, the Rpa1-D223Y mutation provokes telomere shortening. Here, we show that this mutation impairs lagging-strand telomere replication and leads to the accumulation of secondary structures and recruitment of the homologous recombination factor Rad52. The presence of these secondary DNA structures correlates with reduced association of shelterin subunits Pot1 and Ccq1 at telomeres. Strikingly, heterologous expression of the budding yeast Pif1 known to efficiently unwind G-quadruplex rescues all the telomeric defects of the D223Y cells. Furthermore, in vitro data show that the identical D to Y mutation in human RPA specifically affects its ability to bind G-quadruplex. We propose that RPA prevents the formation of G-quadruplex structures at lagging-strand telomeres to promote shelterin association and facilitate telomerase action at telomeres.

Swi1Timeless Prevents Repeat Instability at Fission Yeast Telomeres.

Genomic instability associated with DNA replication stress is linked to cancer and genetic pathologies in humans. If not properly regulated, replication stress, such as fork stalling and collapse, can be induced at natural replication impediments present throughout the genome. The fork protection complex (FPC) is thought to play a critical role in stabilizing stalled replication forks at several known replication barriers including eukaryotic rDNA genes and the fission yeast mating-type locus. However, little is known about the role of the FPC at other natural impediments including telomeres. Telomeres are considered to be difficult to replicate due to the presence of repetitive GT-rich sequences and telomere-binding proteins. However, the regulatory mechanism that ensures telomere replication is not fully understood. Here, we report the role of the fission yeast Swi1(Timeless), a subunit of the FPC, in telomere replication. Loss of Swi1 causes telomere shortening in a telomerase-independent manner. Our epistasis analyses suggest that heterochromatin and telomere-binding proteins are not major impediments for telomere replication in the absence of Swi1. Instead, repetitive DNA sequences impair telomere integrity in swi1Δ mutant cells, leading to the loss of repeat DNA. In the absence of Swi1, telomere shortening is accompanied with an increased recruitment of Rad52 recombinase and more frequent amplification of telomere/subtelomeres, reminiscent of tumor cells that utilize the alternative lengthening of telomeres pathway (ALT) to maintain telomeres. These results suggest that Swi1 ensures telomere replication by suppressing recombination and repeat instability at telomeres. Our studies may also be relevant in understanding the potential role of Swi1(Timeless) in regulation of telomere stability in cancer cells.

Tpz1-Ccq1 and Tpz1-Poz1 interactions within fission yeast shelterin modulate Ccq1 Thr93 phosphorylation and telomerase recruitment.

In both fission yeast and humans, the shelterin complex plays central roles in regulation of telomerase recruitment, protection of telomeres against DNA damage response factors, and formation of heterochromatin at telomeres. While shelterin is essential for limiting activation of the DNA damage checkpoint kinases ATR and ATM at telomeres, these kinases are required for stable maintenance of telomeres. In fission yeast, Rad3ATR and Tel1ATM kinases are redundantly required for telomerase recruitment, since Rad3ATR/Tel1ATM-dependent phosphorylation of the shelterin subunit Ccq1 at Thr93 promotes interaction between Ccq1 and the telomerase subunit Est1. However, it remained unclear how protein-protein interactions within the shelterin complex (consisting of Taz1, Rap1, Poz1, Tpz1, Pot1 and Ccq1) contribute to the regulation of Ccq1 Thr93 phosphorylation and telomerase recruitment. In this study, we identify domains and amino acid residues that are critical for mediating Tpz1-Ccq1 and Tpz1-Poz1 interaction within the fission yeast shelterin complex. Using separation of function Tpz1 mutants that maintain Tpz1-Pot1 interaction but specifically disrupt either Tpz1-Ccq1 or Tpz1-Poz1 interaction, we then establish that Tpz1-Ccq1 interaction promotes Ccq1 Thr93 phosphorylation, telomerase recruitment, checkpoint inhibition and telomeric heterochromatin formation. Furthermore, we demonstrate that Tpz1-Poz1 interaction promotes telomere association of Poz1, and loss of Poz1 from telomeres leads to increases in Ccq1 Thr93 phosphorylation and telomerase recruitment, and telomeric heterochromatin formation defect. In addition, our studies establish that Tpz1-Poz1 and Tpz1-Ccq1 interactions redundantly fulfill the essential telomere protection function of the shelterin complex, since simultaneous loss of both interactions caused immediate loss of cell viability for the majority of cells and generation of survivors with circular chromosomes. Based on these findings, we suggest that the negative regulatory function of Tpz1-Poz1 interaction works upstream of Rad3ATR kinase, while Tpz1-Ccq1 interaction works downstream of Rad3ATR kinase to facilitate Ccq1 Thr93 phosphorylation and telomerase recruitment.

SUMOylation regulates telomere length by targeting the shelterin subunit Tpz1(Tpp1) to modulate shelterin-Stn1 interaction in fission yeast.

Telomeres protect DNA ends of linear eukaryotic chromosomes from degradation and fusion, and ensure complete replication of the terminal DNA through recruitment of telomerase. The regulation of telomerase is a critical area of telomere research and includes cis regulation by the shelterin complex in mammals and fission yeast. We have identified a key component of this regulatory pathway as the SUMOylation [the covalent attachment of a small ubiquitin-like modifier (SUMO) to target proteins] of a shelterin subunit in fission yeast. SUMOylation is known to be involved in the negative regulation of telomere extension by telomerase; however, how SUMOylation limits the action of telomerase was unknown until now. We show that SUMOylation of the shelterin subunit TPP1 homolog in Schizosaccharomyces pombe (Tpz1) on lysine 242 is important for telomere length homeostasis. Furthermore, we establish that Tpz1 SUMOylation prevents telomerase accumulation at telomeres by promoting recruitment of Stn1-Ten1 to telomeres. Our findings provide major mechanistic insights into how the SUMOylation pathway collaborates with shelterin and Stn1-Ten1 complexes to regulate telomere length.

HAATI survivors replace canonical telomeres with blocks of generic heterochromatin.

The notion that telomeres are essential for chromosome linearity stems from the existence of two chief dangers: inappropriate DNA damage response (DDR) reactions that mistake natural chromosome ends for double-strand DNA breaks (DSBs), and the progressive loss of DNA from chromosomal termini due to the end replication problem. Telomeres avert the former peril by binding sequence-specific end-protection factors that control the access of DDR activities. The latter threat is tackled by recruiting telomerase, a reverse transcriptase that uses an integral RNA subunit to template the addition of telomere repeats to chromosome ends. Here we describe an alternative mode of linear chromosome maintenance in which canonical telomeres are superseded by blocks of heterochromatin. We show that in the absence of telomerase, Schizosaccharomyces pombe cells can survive telomere sequence loss by continually amplifying and rearranging heterochromatic sequences. Because the heterochromatin assembly machinery is required for this survival mode, we have termed it 'HAATI' (heterochromatin amplification-mediated and telomerase-independent). HAATI uses the canonical end-protection protein Pot1 (ref. 4) and its interacting partner Ccq1 (ref. 5) to preserve chromosome linearity. The data suggest a model in which Ccq1 is recruited by the amplified heterochromatin and provides an anchor for Pot1, which accomplishes its end-protection function in the absence of its cognate DNA-binding sequence. HAATI resembles the chromosome end-maintenance strategy found in Drosophila melanogaster, which lacks specific telomere sequences but nonetheless assembles terminal heterochromatin structures that recruit end-protection factors. These findings reveal a previously unrecognized mode by which cancer cells might escape the requirement for telomerase activation, and offer a tool for studying genomes that sustain unusually high levels of heterochromatinization.

Telomeres avoid end detection by severing the checkpoint signal transduction pathway.

Telomeres protect the normal ends of chromosomes from being recognized as deleterious DNA double-strand breaks. Recent studies have uncovered an apparent paradox: although DNA repair is prevented, several proteins involved in DNA damage processing and checkpoint responses are recruited to telomeres in every cell cycle and are required for end protection. It is currently not understood how telomeres prevent DNA damage responses from causing permanent cell cycle arrest. Here we show that fission yeast (Schizosaccharomyces pombe) cells lacking Taz1, an orthologue of human TRF1 and TRF2 (ref. 2), recruit DNA repair proteins (Rad22(RAD52) and Rhp51(RAD51), where the superscript indicates the human orthologue) and checkpoint sensors (RPA, Rad9, Rad26(ATRIP) and Cut5/Rad4(TOPBP1)) to telomeres. Despite this, telomeres fail to accumulate the checkpoint mediator Crb2(53BP1) and, consequently, do not activate Chk1-dependent cell cycle arrest. Artificially recruiting Crb2(53BP1) to taz1Δ telomeres results in a full checkpoint response and cell cycle arrest. Stable association of Crb2(53BP1) to DNA double-strand breaks requires two independent histone modifications: H4 dimethylation at lysine 20 (H4K20me2) and H2A carboxy-terminal phosphorylation (γH2A). Whereas γH2A can be readily detected, telomeres lack H4K20me2, in contrast to internal chromosome locations. Blocking checkpoint signal transduction at telomeres requires Pot1 and Ccq1, and loss of either Pot1 or Ccq1 from telomeres leads to Crb2(53BP1) foci formation, Chk1 activation and cell cycle arrest. Thus, telomeres constitute a chromatin-privileged region of the chromosomes that lack essential epigenetic markers for DNA damage response amplification and cell cycle arrest. Because the protein kinases ATM and ATR must associate with telomeres in each S phase to recruit telomerase, exclusion of Crb2(53BP1) has a critical role in preventing telomeres from triggering cell cycle arrest.

Fission yeast shelterin regulates DNA polymerases and Rad3(ATR) kinase to limit telomere extension.

Studies in fission yeast have previously identified evolutionarily conserved shelterin and Stn1-Ten1 complexes, and established Rad3(ATR)/Tel1(ATM)-dependent phosphorylation of the shelterin subunit Ccq1 at Thr93 as the critical post-translational modification for telomerase recruitment to telomeres. Furthermore, shelterin subunits Poz1, Rap1 and Taz1 have been identified as negative regulators of Thr93 phosphorylation and telomerase recruitment. However, it remained unclear how telomere maintenance is dynamically regulated during the cell cycle. Thus, we investigated how loss of Poz1, Rap1 and Taz1 affects cell cycle regulation of Ccq1 Thr93 phosphorylation and telomere association of telomerase (Trt1(TERT)), DNA polymerases, Replication Protein A (RPA) complex, Rad3(ATR)-Rad26(ATRIP) checkpoint kinase complex, Tel1(ATM) kinase, shelterin subunits (Tpz1, Ccq1 and Poz1) and Stn1. We further investigated how telomere shortening, caused by trt1Δ or catalytically dead Trt1-D743A, affects cell cycle-regulated telomere association of telomerase and DNA polymerases. These analyses established that fission yeast shelterin maintains telomere length homeostasis by coordinating the differential arrival of leading (Polε) and lagging (Polα) strand DNA polymerases at telomeres to modulate Rad3(ATR) association, Ccq1 Thr93 phosphorylation and telomerase recruitment.

Differential arrival of leading and lagging strand DNA polymerases at fission yeast telomeres.

To maintain genomic integrity, telomeres must undergo switches from a protected state to an accessible state that allows telomerase recruitment. To better understand how telomere accessibility is regulated in fission yeast, we analysed cell cycle-dependent recruitment of telomere-specific proteins (telomerase Trt1, Taz1, Rap1, Pot1 and Stn1), DNA replication proteins (DNA polymerases, MCM, RPA), checkpoint protein Rad26 and DNA repair protein Nbs1 to telomeres. Quantitative chromatin immunoprecipitation studies revealed that MCM, Nbs1 and Stn1 could be recruited to telomeres in the absence of telomere replication in S-phase. In contrast, Trt1, Pot1, RPA and Rad26 failed to efficiently associate with telomeres unless telomeres are actively replicated. Unexpectedly, the leading strand DNA polymerase epsilon (Polepsilon) arrived at telomeres earlier than the lagging strand DNA polymerases alpha (Polalpha) and delta (Poldelta). Recruitment of RPA and Rad26 to telomeres matched arrival of DNA Polepsilon, whereas S-phase specific recruitment of Trt1, Pot1 and Stn1 matched arrival of DNA Polalpha. Thus, the conversion of telomere states involves an unanticipated intermediate step where lagging strand synthesis is delayed until telomerase is recruited.

A kinase-independent role for the Rad3(ATR)-Rad26(ATRIP) complex in recruitment of Tel1(ATM) to telomeres in fission yeast.

ATM and ATR are two redundant checkpoint kinases essential for the stable maintenance of telomeres in eukaryotes. Previous studies have established that MRN (Mre11-Rad50-Nbs1) and ATRIP (ATR Interacting Protein) interact with ATM and ATR, respectively, and recruit their partner kinases to sites of DNA damage. Here, we investigated how Tel1(ATM) and Rad3(ATR) recruitment to telomeres is regulated in fission yeast. Quantitative chromatin immunoprecipitation (ChIP) assays unexpectedly revealed that the MRN complex could also contribute to the recruitment of Tel1(ATM) to telomeres independently of the previously established Nbs1 C-terminal Tel1(ATM) interaction domain. Recruitment of Tel1(ATM) to telomeres in nbs1-c60Delta cells, which lack the C-terminal 60 amino acid Tel1(ATM) interaction domain of Nbs1, was dependent on Rad3(ATR)-Rad26(ATRIP), but the kinase domain of Rad3(ATR) was dispensable. Thus, our results establish that the Rad3(ATR)-Rad26(ATRIP) complex contributes to the recruitment of Tel1(ATM) independently of Rad3(ATR) kinase activity, by a mechanism redundant with the Tel1(ATM) interaction domain of Nbs1. Furthermore, we found that the N-terminus of Nbs1 contributes to the recruitment of Rad3(ATR)-Rad26(ATRIP) to telomeres. In response to replication stress, mammalian ATR-ATRIP also contributes to ATM activation by a mechanism that is dependent on the MRN complex but independent of the C-terminal ATM interaction domain of Nbs1. Since telomere protection and DNA damage response mechanisms are very well conserved between fission yeast and mammalian cells, mammalian ATR-ATRIP may also contribute to the recruitment of ATM to telomeres and to sites of DNA damage independently of ATR kinase activity.

Comprehensive mutational analysis of the checkpoint signaling function of Rpa1/Ssb1 in fission yeast.

Replication protein A (RPA) is a heterotrimeric complex and the major single-strand DNA (ssDNA) binding protein in eukaryotes. It plays important roles in DNA replication, repair, recombination, telomere maintenance, and checkpoint signaling. Because RPA is essential for cell survival, understanding its checkpoint signaling function in cells has been challenging. Several RPA mutants have been reported previously in fission yeast. None of them, however, has a defined checkpoint defect. A separation-of-function mutant of RPA, if identified, would provide significant insights into the checkpoint initiation mechanisms. We have explored this possibility and carried out an extensive genetic screening for Rpa1/Ssb1, the large subunit of RPA in fission yeast, looking for mutants with defects in checkpoint signaling. This screen has identified twenty-five primary mutants that are sensitive to genotoxins. Among these mutants, two have been confirmed partially defective in checkpoint signaling primarily at the replication fork, not the DNA damage site. The remaining mutants are likely defective in other functions such as DNA repair or telomere maintenance. Our screened mutants, therefore, provide a valuable tool for future dissection of the multiple functions of RPA in fission yeast. AUTHOR SUMMARY: Originally discovered as a protein required for replication of simian virus SV40 DNA, replication protein A is now known to function in DNA replication, repair, recombination, telomere maintenance, and checkpoint signaling in all eukaryotes. The protein is a complex of three subunits and the two larger ones are essential for cell growth. This essential function however complicates the studies in living cells, and for this reason, its checkpoint function remains to be fully understood. We have carried out an genetic screening of the largest subunit of this protein in fission yeast, aiming to find a non-lethal mutant that lacks the checkpoint function. This extensive screen has uncovered two mutants with a partial defect in checkpoint signaling when DNA replication is arrested. Surprisingly, although the two mutants also have a defect in DNA repair, their checkpoint signaling remains largely functional in the presence of DNA damage. We have also uncovered twenty-three mutants with defects in DNA repair or telomere maintenance, but not checkpoint signaling. Therefore, the non-lethal mutants uncovered by this study provide a valuable tool for dissecting the multiple functions of this biologically important protein in fission yeast.

Fission yeast Tel1(ATM) and Rad3(ATR) promote telomere protection and telomerase recruitment.

The checkpoint kinases ATM and ATR are redundantly required for maintenance of stable telomeres in diverse organisms, including budding and fission yeasts, Arabidopsis, Drosophila, and mammals. However, the molecular basis for telomere instability in cells lacking ATM and ATR has not yet been elucidated fully in organisms that utilize both the telomere protection complex shelterin and telomerase to maintain telomeres, such as fission yeast and humans. Here, we demonstrate by quantitative chromatin immunoprecipitation (ChIP) assays that simultaneous loss of Tel1(ATM) and Rad3(ATR) kinases leads to a defect in recruitment of telomerase to telomeres, reduced binding of the shelterin complex subunits Ccq1 and Tpz1, and increased binding of RPA and homologous recombination repair factors to telomeres. Moreover, we show that interaction between Tpz1-Ccq1 and telomerase, thought to be important for telomerase recruitment to telomeres, is disrupted in tel1Delta rad3Delta cells. Thus, Tel1(ATM) and Rad3(ATR) are redundantly required for both protection of telomeres against recombination and promotion of telomerase recruitment. Based on our current findings, we propose the existence of a regulatory loop between Tel1(ATM)/Rad3(ATR) kinases and Tpz1-Ccq1 to ensure proper protection and maintenance of telomeres in fission yeast.

Roles of heterochromatin and telomere proteins in regulation of fission yeast telomere recombination and telomerase recruitment.

When the telomerase catalytic subunit (Trt1/TERT) is deleted, a majority of fission yeast cells survives by circularizing chromosomes. Alternatively, a small minority survives by maintaining telomeric repeats through recombination among telomeres. The recombination-based telomere maintenance in trt1Delta cells is inhibited by the telomere protein Taz1. In addition, catalytically inactive full-length Trt1 (Trt1-CI) and truncated Trt1 lacking the T-motif and reverse transcriptase (RT) domain (Trt1-DeltaT/RT) can strongly inhibit recombination-based survival. Here, we investigated the effects of deleting the heterochromatin proteins Swi6 (HP1 ortholog) and Clr4 (Suv39 family of histone methyltransferases) and the telomere capping complex subunits Poz1 and Ccq1 on Taz1- and Trt1-dependent telomere recombination inhibition. The ability of Taz1 to inhibit telomere recombination did not require Swi6, Clr4, Poz1, or Ccq1. Although Swi6, Clr4, and Poz1 were dispensable for the inhibition of telomere recombination by Trt1-CI, Ccq1 was required for efficient telomere recruitment of Trt1 and Trt1-CI-dependent inhibition of telomere recombination. We also found that Swi6, Clr4, Ccq1, the checkpoint kinase Rad3 (ATR ortholog), and the telomerase regulatory subunit Est1 are all required for Trt1-DeltaT/RT to inhibit telomere recombination. However, because loss of Swi6, Clr4, Rad3, Ccq1, or Est1 did not significantly alter the recruitment efficiency of Trt1-DeltaT/RT to telomeres, these factors are likely to enhance the ability of Trt1-DeltaT/RT to inhibit recombination-based survival by contributing to the negative regulation of telomere recombination.

Protection and replication of telomeres in fission yeast.

Telomeres, the natural ends of linear chromosomes, must be protected and completely replicated to guarantee genomic stability in eukaryotic cells. However, the protected state of telomeres is not compatible with recruitment of telomerase, an enzyme responsible for extending telomeric G-rich repeats during S-phase; thus, telomeres must undergo switches from a protected state to an accessible state during the cell cycle. In this minireview, we will summarize recent advances in our understanding of proteins involved in the protection and replication of telomeres, and the way these factors are dynamically recruited to telomeres during the cell cycle. We will focus mainly on recent results from fission yeast Schizosaccharomyces pombe, and compare them with results from budding yeast Saccharomyces cerevisiae and mammalian cell studies. In addition, a model for the way in which fission yeast cells replicate telomeres will be presented.

Rad22Rad52-dependent repair of ribosomal DNA repeats cleaved by Slx1-Slx4 endonuclease.

Slx1 and Slx4 are subunits of a structure-specific DNA endonuclease that is found in Saccharomyces cerevisiae, Schizosaccharomyces pombe, and other eukaryotic species. It is thought to initiate recombination events or process recombination structures that occur during the replication of the tandem repeats of the ribosomal DNA (rDNA) locus. Here, we present evidence that fission yeast Slx1-Slx4 initiates homologous recombination events in the rDNA repeats that are processed by a mechanism that requires Rad22 (Rad52 homologue) but not Rhp51 (Rad51 homologue). Slx1 is required to generate approximately 50% of the spontaneous Rad22 DNA repair foci that occur in cycling cells. Most of these foci colocalize with the nucleolus, which contains the rDNA repeats. The increased fork pausing at the replication fork barriers in the rDNA repeats in a strain that lacks Rqh1 DNA helicase is further increased by expression of a dominant negative form of Slx1. These data suggest that Slx1-Slx4 cleaves paused replication forks in the rDNA, leading to Rad22-dependent homologous recombination that is used to maintain rDNA copy number.

Tpz1TPP1 prevents telomerase activation and protects telomeres by modulating the Stn1-Ten1 complex in fission yeast.

In both mammalian and fission yeast cells, conserved shelterin and CST (CTC1-STN1-TEN1) complexes play critical roles in protection of telomeres and regulation of telomerase, an enzyme required to overcome the end replication problem. However, molecular details that govern proper coordination among shelterin, CST, and telomerase have not yet been fully understood. Here, we establish a conserved SWSSS motif, located adjacent to the Lys242 SUMOylation site in the fission yeast shelterin subunit Tpz1, as a new functional regulatory element for telomere protection and telomere length homeostasis. The SWSSS motif works redundantly with Lys242 SUMOylation to promote binding of Stn1-Ten1 at telomere and sub-telomere regions to protect against single-strand annealing (SSA)-dependent telomere fusions, and to prevent telomerase accumulation at telomeres. In addition, we provide evidence that the SWSSS motif defines an unanticipated role of Tpz1 in limiting telomerase activation at telomeres to prevent uncontrolled telomere elongation.

The NuA4 acetyltransferase and histone H4 acetylation promote replication recovery after topoisomerase I-poisoning.

BACKGROUND: Histone acetylation plays an important role in DNA replication and repair because replicating chromatin is subject to dynamic changes in its structures. However, its precise mechanism remains elusive. In this report, we describe roles of the NuA4 acetyltransferase and histone H4 acetylation in replication fork protection in the fission yeast Schizosaccharomyces pombe. RESULTS: Downregulation of NuA4 subunits renders cells highly sensitive to camptothecin, a compound that induces replication fork breakage. Defects in NuA4 function or mutations in histone H4 acetylation sites lead to impaired recovery of collapsed replication forks and elevated levels of Rad52 DNA repair foci, indicating the role of histone H4 acetylation in DNA replication and fork repair. We also show that Vid21 interacts with the Swi1-Swi3 replication fork protection complex and that Swi1 stabilizes Vid21 and promotes efficient histone H4 acetylation. Furthermore, our genetic analysis demonstrates that loss of Swi1 further sensitizes NuA4 and histone H4 mutant cells to replication fork breakage. CONCLUSION: Considering that Swi1 plays a critical role in replication fork protection, our results indicate that NuA4 and histone H4 acetylation promote repair of broken DNA replication forks.

A tel2 Mutation That Destabilizes the Tel2-Tti1-Tti2 Complex Eliminates Rad3ATR Kinase Signaling in the DNA Replication Checkpoint and Leads to Telomere Shortening in Fission Yeast.

In response to perturbed DNA replication, ATR (ataxia telangiectasia and Rad3-related) kinase is activated to initiate the checkpoint signaling necessary for maintaining genome integrity and cell survival. To better understand the signaling mechanism, we carried out a large-scale genetic screen in fission yeast looking for mutants with enhanced sensitivity to hydroxyurea. From a collection of ∼370 primary mutants, we found a few mutants in which Rad3 (ATR ortholog)-mediated phospho-signaling was significantly compromised. One such mutant carried an uncharacterized mutation in tel2, a gene encoding an essential and highly conserved eukaryotic protein. Previous studies in various biological models have shown that Tel2 mainly functions in Tel2-Tti1-Tti2 (TTT) complex that regulates the steady-state levels of all phosphatidylinositol 3-kinase-like protein kinases, including ATR. We show here that although the levels of Rad3 and Rad3-mediated phospho-signaling in DNA damage checkpoint were moderately reduced in the tel2 mutant, the phospho-signaling in the DNA replication checkpoint was almost completely eliminated. In addition, the tel2 mutation caused telomere shortening. Since the interactions of Tel2 with Tti1 and Tti2 were significantly weakened by the mutation, destabilization of the TTT complex likely contributes to the observed checkpoint and telomere defects.

Cooperative control of Crb2 by ATM family and Cdc2 kinases is essential for the DNA damage checkpoint in fission yeast.

The cellular responses to double-stranded breaks (DSBs) typically involve the extensive accumulation of checkpoint proteins in chromatin surrounding the damaged DNA. One well-characterized example involves the checkpoint protein Crb2 in the fission yeast Schizosaccharomyces pombe. The accumulation of Crb2 at DSBs requires the C-terminal phosphorylation of histone H2A (known as gamma-H2A) by ATM family kinases in chromatin surrounding the break. It also requires the constitutive methylation of histone H4 on lysine-20 (K20). Interestingly, neither type of histone modification is essential for the Crb2-dependent checkpoint response. However, H4-K20 methylation is essential in a crb2-T215A strain that lacks a cyclin-dependent kinase phosphorylation site in Crb2. Here we explain this genetic interaction by describing a previously overlooked effect of the crb2-T215A mutation. We show that crb2-T215A cells are able to initiate but not sustain a checkpoint response. We also report that gamma-H2A is essential for the DNA damage checkpoint in crb2-T215A cells. Importantly, we show that inactivation of Cdc2 in gamma-H2A-defective cells impairs Crb2-dependent signaling to the checkpoint kinase Chk1. These findings demonstrate that full Crb2 activity requires phosphorylation of threonine-215 by Cdc2. This regulation of Crb2 is independent of the histone modifications that are required for the hyperaccumulation of Crb2 at DSBs.