Detection of cellular G-quadruplex by using a loop structure as a structural determinant.

G-quadrupex is now known to play crucial roles in various biological reactions. However, direct evidence for its presence in cells has been limited, due to the lack of versatile and non-biased methodology. We use Rif1 binding sites on the fission yeast genome, which has been shown to adopt G4 structures, as a model to prove that Rif1 BS indeed adopt G4 structure in cells. We take advantage of the presence of a single-stranded loop in the G4 structure. Rif1BS is unique in that they contain unusually long loop sequences, and we replace them with a 18 bp I-SceI restriction site. We show in vitro that I-SceI in the loop is not cleaved when G4 is formed on duplex Rif1BS DNA, but is cleaved when G4 is not formed due to a mutation in the G-tracts. This is observed both heat-induced and transcription-induced G4 structure, and gives proof of evidence for this procedure. We apply this strategy for detection of a G4 structure at the same Rif1BS in fission yeast cells. We present evidence that in vivo cleavage of I-SceI can be a measure for the presence of G4 at the target sequence in cells as well. The method described here gives a platform strategy for genome-wide analyses of cellular G4 and their dynamic formation and disruption.

Establishment of expression-state boundaries by Rif1 and Taz1 in fission yeast.

The Shelterin component Rif1 has emerged as a global regulator of the replication-timing program in all eukaryotes examined to date, possibly by modulating the 3D-organization of the genome. In fission yeast a second Shelterin component, Taz1, might share similar functions. Here, we identified unexpected properties for Rif1 and Taz1 by conducting high-throughput genetic screens designed to identify cis- and trans-acting factors capable of creating heterochromatin-euchromatin boundaries in fission yeast. The preponderance of cis-acting elements identified in the screens originated from genomic loci bound by Taz1 and associated with origins of replication whose firing is repressed by Taz1 and Rif1. Boundary formation and gene silencing by these elements required Taz1 and Rif1 and coincided with altered replication timing in the region. Thus, small chromosomal elements sensitive to Taz1 and Rif1 (STAR) could simultaneously regulate gene expression and DNA replication over a large domain, at the edge of which they established a heterochromatin-euchromatin boundary. Taz1, Rif1, and Rif1-associated protein phosphatases Sds21 and Dis2 were each sufficient to establish a boundary when tethered to DNA. Moreover, efficient boundary formation required the amino-terminal domain of the Mcm4 replicative helicase onto which the antagonistic activities of the replication-promoting Dbf4-dependent kinase and Rif1-recruited phosphatases are believed to converge to control replication origin firing. Altogether these observations provide an insight into a coordinated control of DNA replication and organization of the genome into expression domains.

Rif1 is a global regulator of timing of replication origin firing in fission yeast.

One of the long-standing questions in eukaryotic DNA replication is the mechanisms that determine where and when a particular segment of the genome is replicated. Cdc7/Hsk1 is a conserved kinase required for initiation of DNA replication and may affect the site selection and timing of origin firing. We identified rif1Δ, a null mutant of rif1(+), a conserved telomere-binding factor, as an efficient bypass mutant of fission yeast hsk1. Extensive deregulation of dormant origins over a wide range of the chromosomes occurs in rif1Δ in the presence or absence of hydroxyurea (HU). At the same time, many early-firing, efficient origins are suppressed or delayed in firing timing in rif1Δ. Rif1 binds not only to telomeres, but also to many specific locations on the arm segments that only partially overlap with the prereplicative complex assembly sites, although Rif1 tends to bind in the vicinity of the late/dormant origins activated in rif1Δ. The binding to the arm segments occurs through M to G1 phase in a manner independent of Taz1 and appears to be essential for the replication timing program during the normal cell cycle. Our data demonstrate that Rif1 is a critical determinant of the origin activation program on the fission yeast chromosomes.

Molecular architecture of G-quadruplex structures generated on duplex Rif1-binding sequences.

G-quadruplexes (G4s) are four-stranded DNA structures comprising stacks of four guanines, are prevalent in genomes, and have diverse biological functions in various chromosomal structures. A conserved protein, Rap1-interacting factor 1 (Rif1) from fission yeast (Schizosaccharomyces pombe), binds to Rif1-binding sequence (Rif1BS) and regulates DNA replication timing. Rif1BS is characterized by the presence of multiple G-tracts, often on both strands, and their unusual spacing. Although previous studies have suggested generation of G4-like structures on duplex Rif1BS, its precise molecular architecture remains unknown. Using gel-shift DNA binding assays and DNA footprinting with various nuclease probes, we show here that both of the Rif1BS strands adopt specific higher-order structures upon heat denaturation. We observed that the structure generated on the G-strand is consistent with a G4 having unusually long loop segments and that the structure on the complementary C-strand does not have an intercalated motif (i-motif). Instead, we found that the formation of the C-strand structure depends on the G4 formation on the G-strand. Thus, the higher-order structure generated at Rif1BS involved both DNA strands, and in some cases, G4s may form on both of these strands. The presence of multiple G-tracts permitted the formation of alternative structures when some G-tracts were mutated or disrupted by deazaguanine replacement, indicating the robust nature of DNA higher-order structures generated at Rif1BS. Our results provide general insights into DNA structures generated at G4-forming sequences on duplex DNA.

Replication termination at eukaryotic chromosomes is mediated by Top2 and occurs at genomic loci containing pausing elements.

Chromosome replication initiates at multiple replicons and terminates when forks converge. In E. coli, the Tus-TER complex mediates polar fork converging at the terminator region, and aberrant termination events challenge chromosome integrity and segregation. Since in eukaryotes, termination is less characterized, we used budding yeast to identify the factors assisting fork fusion at replicating chromosomes. Using genomic and mechanistic studies, we have identified and characterized 71 chromosomal termination regions (TERs). TERs contain fork pausing elements that influence fork progression and merging. The Rrm3 DNA helicase assists fork progression across TERs, counteracting the accumulation of X-shaped structures. The Top2 DNA topoisomerase associates at TERs in S phase, and G2/M facilitates fork fusion and prevents DNA breaks and genome rearrangements at TERs. We propose that in eukaryotes, replication fork barriers, Rrm3, and Top2 coordinate replication fork progression and fusion at TERs, thus counteracting abnormal genomic transitions.

Temporal and spatial regulation of eukaryotic DNA replication: from regulated initiation to genome-scale timing program.

Replication origins are where pre-replication complexes are assembled during G1 phase. However, only a subset of the origins is actually "fired" to initiate DNA synthesis during S phase. Whereas factors involved in these steps are relatively well understood now, the mechanisms behind the origin specification, the choice of origins to be fired and determination of their timing are still under active investigation. Recent data show that the origin positions as well as the selection of those to be fired may be determined by multiple factors including sequences, chromatin context, epigenetic information, and some specific genomic features, but that the choice is surprisingly plastic and opportunistic. Timing regulation of firing, on the other hand, appears to be related to cell type-specific intrinsic chromatin architecture in nuclei. The conserved Rif1 protein appears to be a major global regulator of the genome-wide replication timing. Replication timing is regulated also by other factors including checkpoint signals, local chromatin structures, timing and quantity of pre-RC formation, and availability of limiting initiation factors.

Rif1 regulates the replication timing domains on the human genome.

DNA replication is spatially and temporally regulated during S-phase. DNA replication timing is established in early-G1-phase at a point referred to as timing decision point. However, how the genome-wide replication timing domains are established is unknown. Here, we show that Rif1 (Rap1-interacting-factor-1), originally identified as a telomere-binding factor in yeast, is a critical determinant of the replication timing programme in human cells. Depletion of Rif1 results in specific loss of mid-S replication foci profiles, stimulation of initiation events in early-S-phase and changes in long-range replication timing domain structures. Analyses of replication timing show replication of sequences normally replicating early is delayed, whereas that normally replicating late is advanced, suggesting that replication timing regulation is abrogated in the absence of Rif1. Rif1 tightly binds to nuclear-insoluble structures at late-M-to-early-G1 and regulates chromatin-loop sizes. Furthermore, Rif1 colocalizes specifically with the mid-S replication foci. Thus, Rif1 establishes the mid-S replication domains that are restrained from being activated at early-S-phase. Our results indicate that Rif1 plays crucial roles in determining the replication timing domain structures in human cells through regulating higher-order chromatin architecture.

Homologous recombination contributes to the repair of acetaldehyde-induced DNA damage.

Acetaldehyde, a chemical that can cause DNA damage and contribute to cancer, is prevalently present in our environment, e.g. in alcohol, tobacco, and food. Although aldehyde potentially promotes crosslinking reactions among biological substances including DNA, RNA, and protein, it remains unclear what types of DNA damage are caused by acetaldehyde and how they are repaired. In this study, we explored mechanisms involved in the repair of acetaldehyde-induced DNA damage by examining the cellular sensitivity to acetaldehyde in the collection of human TK6 mutant deficient in each genome maintenance system. Among the mutants, mismatch repair mutants did not show hypersensitivity to acetaldehyde, while mutants deficient in base and nucleotide excision repair pathways or homologous recombination (HR) exhibited higher sensitivity to acetaldehyde than did wild-type cells. We found that acetaldehyde-induced RAD51 foci representing HR intermediates were prolonged in HR-deficient cells. These results indicate a pivotal role of HR in the repair of acetaldehyde-induced DNA damage. These results suggest that acetaldehyde causes complex DNA damages that require various types of repair pathways. Mutants deficient in the removal of protein adducts from DNA ends such as TDP1-/- and TDP2-/- cells exhibited hypersensitivity to acetaldehyde. Strikingly, the double mutant deficient in both TDP1 and RAD54 showed similar sensitivity to each single mutant. This epistatic relationship between TDP1-/- and RAD54-/- suggests that the protein-DNA adducts generated by acetaldehyde need to be removed for efficient repair by HR. Our study would help understand the molecular mechanism of the genotoxic and mutagenic effects of acetaldehyde.

Aberrant association of chromatin with nuclear periphery induced by Rif1 leads to mitotic defect.

The architecture and nuclear location of chromosomes affect chromatin events. Rif1, a crucial regulator of replication timing, recognizes G-quadruplex and inhibits origin firing over the 50-100-kb segment in fission yeast, Schizosaccharomyces pombe, leading us to postulate that Rif1 may generate chromatin higher order structures inhibitory for initiation. However, the effects of Rif1 on chromatin localization in nuclei have not been known. We show here that Rif1 overexpression causes growth inhibition and eventually, cell death in fission yeast. Chromatin-binding activity of Rif1, but not recruitment of phosphatase PP1, is required for growth inhibition. Overexpression of a PP1-binding site mutant of Rif1 does not delay the S-phase, but still causes cell death, indicating that cell death is caused not by S-phase problems but by issues in other phases of the cell cycle, most likely the M-phase. Indeed, Rif1 overexpression generates cells with unequally segregated chromosomes. Rif1 overexpression relocates chromatin near nuclear periphery in a manner dependent on its chromatin-binding ability, and this correlates with growth inhibition. Thus, coordinated progression of S- and M-phases may require regulated Rif1-mediated chromatin association with the nuclear periphery.

S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex.

The checkpoint regulatory mechanism has an important role in maintaining the integrity of the genome. This is particularly important in S phase of the cell cycle, when genomic DNA is most susceptible to various environmental hazards. When chemical agents damage DNA, activation of checkpoint signalling pathways results in a temporary cessation of DNA replication. A replication-pausing complex is believed to be created at the arrested forks to activate further checkpoint cascades, leading to repair of the damaged DNA. Thus, checkpoint factors are thought to act not only to arrest replication but also to maintain a stable replication complex at replication forks. However, the molecular mechanism coupling checkpoint regulation and replication arrest is unknown. Here we demonstrate that the checkpoint regulatory proteins Tof1 and Mrc1 interact directly with the DNA replication machinery in Saccharomyces cerevisiae. When hydroxyurea blocks chromosomal replication, this assembly forms a stable pausing structure that serves to anchor subsequent DNA repair events.

Both a Unique Motif at the C Terminus and an N-Terminal HEAT Repeat Contribute to G-Quadruplex Binding and Origin Regulation by the Rif1 Protein.

Rif1 is a key factor for spatiotemporal regulation of DNA replication. Rif1 suppresses origin firing in the mid-late replication domains by generating replication-suppressive chromatin architecture and by recruiting a protein phosphatase. In fission yeast, the function of Hsk1, a kinase important for origin firing, can be bypassed by rif1Δ due to the loss of origin suppression. Rif1 specifically binds to G-quadruplex (G4) in vitro Here, we show both conserved N-terminal HEAT repeats and C-terminal nonconserved segments are required for origin suppression. The N-terminal 444 amino acids and the C-terminal 229 amino acids can each mediate specific G4 binding, although high-affinity G4 binding requires the presence of both N- and C-terminal segments. The C-terminal 91 amino acids, although not able to bind to G4, can form a multimer. Furthermore, genetic screening led to identification of two classes of rif1 point mutations that can bypass Hsk1, one that fails to bind to chromatin and one that binds to chromatin. These results illustrate functional domains of Rif1 and indicate importance of both the N-terminal HEAT repeat segment and C-terminal G4 binding/oligomerization domain as well as other functionally unassigned segments of Rif1 in regulation of origin firing.

Rif1 promotes association of G-quadruplex (G4) by its specific G4 binding and oligomerization activities.

Rif1 is a conserved protein regulating replication timing and binds preferentially to the vicinity of late-firing/dormant origins in fission yeast. The Rif1 binding sites on the fission yeast genome have an intrinsic potential to generate G-quadruplex (G4) structures to which purified Rif1 preferentially binds. We previously proposed that Rif1 generates chromatin architecture that may determine replication timing by facilitating the chromatin loop formation. Here, we conducted detailed biochemical analyses on Rif1 and its G4 binding. Rif1 prefers sequences containing long stretches of guanines and binds preferentially to the multimeric G4 of parallel or hybrid/mix topology. Rif1 forms oligomers and binds simultaneously to multiple G4. We present a model on how Rif1 may facilitate the formation of chromatin architecture through its G4 binding and oligomerization properties.

Telomere-binding factors in the regulation of DNA replication.

Recent studies have indicated new roles for telomere-binding factors in the regulation of DNA replication, not only at the telomeres but also at the arm regions of the chromosome. Among these factors, Rif1, a conserved protein originally identified in yeasts as a telomere regulator, plays a major role in the spatiotemporal regulation of DNA replication during S phase. Its ability to interact with phosphatases and to create specific higher-order chromatin structures is central to the mechanism by which Rif1 exerts this function. In this review, we discuss recent progress in elucidating the roles of Rif1 and other telomere-binding factors in the regulation of chromosome events occurring at locations other than telomeres.

Checkpoint-Independent Regulation of Origin Firing by Mrc1 through Interaction with Hsk1 Kinase.

Mrc1 is a conserved checkpoint mediator protein that transduces the replication stress signal to the downstream effector kinase. The loss of mrc1 checkpoint activity results in the aberrant activation of late/dormant origins in the presence of hydroxyurea. Mrc1 was also suggested to regulate orders of early origin firing in a checkpoint-independent manner, but its mechanism was unknown. Here we identify HBS (Hsk1 bypass segment) on Mrc1. An ΔHBS mutant does not activate late/dormant origin firing in the presence of hydroxyurea but causes the precocious and enhanced activation of weak early-firing origins during normal S-phase progression and bypasses the requirement for Hsk1 for growth. This may be caused by the disruption of intramolecular binding between HBS and NTHBS (N-terminal target of HBS). Hsk1 binds to Mrc1 through HBS and phosphorylates a segment adjacent to NTHBS, disrupting the intramolecular interaction. We propose that Mrc1 exerts a "brake" on initiation (through intramolecular interactions) and that this brake can be released (upon the loss of intramolecular interactions) by either the Hsk1-mediated phosphorylation of Mrc1 or the deletion of HBS (or a phosphomimic mutation of putative Hsk1 target serine/threonine), which can bypass the function of Hsk1 for growth. The brake mechanism may explain the checkpoint-independent regulation of early origin firing in fission yeast.

Different requirements for the association of ATR-ATRIP and 9-1-1 to the stalled replication forks.

DNA replication checkpoint, a surveillance mechanism for S-phase progression, plays a crucial role for the maintenance of genome integrity. A variety of factors have been characterized to be involved in the checkpoint signal transduction. Rpa, a single strand DNA binding protein, was found to be responsible for forming a structure that is recognized by checkpoint sensors and then emits the initial signal for the activation of DNA damage checkpoint. Here we use a mutant of rpa1 gene, rfa1-t11, that has defects in recruiting checkpoint sensor proteins to the site of double strand break, to examine the mutant's effects on the activation of DNA replication checkpoint. We found that the mutant cells activated DNA replication checkpoint normally and showed no defects in recruiting ATR-ATRIP, a major sensor complex that is essential for DNA replication/damage checkpoint, to the site of stalled forks. In contrast, the mutant was defective in recruiting 9-1-1 complex, another sensor complex that functions in DNA damage checkpoint signal transduction, to the stalled forks. Moreover we found that sensitivity for HU obviously appeared in rfa1-t11 mutant when Mrc1 was deleted, while deletion of Rad9, an adaptor specific for damage checkpoint, had subtle effect. These data strongly suggest that rfa1-t11 mutant was mainly defective for activating DNA damage checkpoint and molecular requirement for the recruitment of ATR-ATRIP and 9-1-1 to the stressed forks may be different.

Rif1 binds to G quadruplexes and suppresses replication over long distances.

Rif1 regulates replication timing and repair of double-strand DNA breaks. Using a chromatin immunoprecipitation-sequencing method, we identified 35 high-affinity Rif1-binding sites in fission yeast chromosomes. Binding sites tended to be located near dormant origins and to contain at least two copies of a conserved motif, CNWWGTGGGGG. Base substitution within these motifs resulted in complete loss of Rif1 binding and in activation of late-firing or dormant origins located up to 50 kb away. We show that Rif1-binding sites adopt G quadruplex-like structures in vitro, in a manner dependent on the conserved sequence and on other G tracts, and that purified Rif1 preferentially binds to this structure. These results suggest that Rif1 recognizes and binds G quadruplex-like structures at selected intergenic regions, thus generating local chromatin structures that may exert long-range suppressive effects on origin firing.

Regulation of DNA replication timing on human chromosome by a cell-type specific DNA binding protein SATB1.

BACKGROUND: Replication timing of metazoan DNA during S-phase may be determined by many factors including chromosome structures, nuclear positioning, patterns of histone modifications, and transcriptional activity. It may be determined by Mb-domain structures, termed as "replication domains", and recent findings indicate that replication timing is under developmental and cell type-specific regulation. METHODOLOGY/PRINCIPAL FINDINGS: We examined replication timing on the human 5q23/31 3.5-Mb segment in T cells and non-T cells. We used two independent methods to determine replication timing. One is quantification of nascent replicating DNA in cell cycle-fractionated stage-specific S phase populations. The other is FISH analyses of replication foci. Although the locations of early- and late-replicating domains were common between the two cell lines, the timing transition region (TTR) between early and late domains were offset by 200-kb. We show that Special AT-rich sequence Binding protein 1 (SATB1), specifically expressed in T-cells, binds to the early domain immediately adjacent to TTR and delays the replication timing of the TTR. Measurement of the chromosome copy number along the TTR during synchronized S phase suggests that the fork movement may be slowed down by SATB1. CONCLUSIONS: Our results reveal a novel role of SATB1 in cell type-specific regulation of replication timing along the chromosome.

Mrc1 marks early-firing origins and coordinates timing and efficiency of initiation in fission yeast.

How early- and late-firing origins are selected on eukaryotic chromosomes is largely unknown. Here, we show that Mrc1, a conserved factor required for stabilization of stalled replication forks, selectively binds to the early-firing origins in a manner independent of Cdc45 and Hsk1 kinase in the fission yeast Schizosaccharomyces pombe. In mrc1Δ cells (and in swi1Δ cells to some extent), efficiency of firing is stimulated, and its timing is advanced selectively at those origins that are normally bound by Mrc1. In contrast, the late or inefficient origins which are not bound by Mrc1 are not activated in mrc1Δ cells. The enhanced firing and precocious Cdc45 loading at Mrc1-bound early-firing origins are not observed in a checkpoint mutant of mrc1, suggesting that non-checkpoint function is involved in maintaining the normal program of early-firing origins. We propose that prefiring binding of Mrc1 is an important marker of early-firing origins which are precociously activated by the absence of this protein.

Multiple pathways can bypass the essential role of fission yeast Hsk1 kinase in DNA replication initiation.

Cdc7/Hsk1 is a conserved kinase required for initiation of DNA replication that potentially regulates timing and locations of replication origin firing. Here, we show that viability of fission yeast hsk1Δ cells can be restored by loss of mrc1, which is required for maintenance of replication fork integrity, by cds1Δ, or by a checkpoint-deficient mutant of mrc1. In these mutants, normally inactive origins are activated in the presence of hydroxyurea and binding of Cdc45 to MCM is stimulated. mrc1Δ bypasses hsk1Δ more efficiently because of its checkpoint-independent inhibitory functions. Unexpectedly, hsk1Δ is viable at 37°C. More DNA is synthesized, and some dormant origins fire in the presence of hydroxyurea at 37°C. Furthermore, hsk1Δ bypass strains grow poorly at 25°C compared with higher temperatures. Our results show that Hsk1 functions for DNA replication can be bypassed by different genetic backgrounds as well as under varied physiological conditions, providing additional evidence for plasticity of the replication program in eukaryotes.

Hsk1 kinase and Cdc45 regulate replication stress-induced checkpoint responses in fission yeast.

In fission yeast, replication fork arrest activates the replication checkpoint effector kinase Cds1(Chk2/Rad53) through the Rad3(ATR/Mec1)-Mrc1(Claspin) pathway. Hsk1, the Cdc7 homologue of fission yeast required for efficient initiation of DNA replication, is also required for Cds1 activation. Hsk1 kinase activity is required for induction and maintenance of Mrc1 hyperphosphorylation, which is induced by replication fork block and mediated by Rad3. Rad3 kinase activity does not change in an hsk1 temperature-sensitive mutant, and Hsk1 kinase activity is not affected by rad3 mutation. Hsk1 kinase vigorously phosphorylates Mrc1 in vitro, predominantly at non-SQ/TQ sites, but this phosphorylation does not seem to affect the Rad3 action on Mrc1. Interestingly, the replication stress-induced activation of Cds1 and hyperphosphorylation of Mrc1 is almost completely abrogated in an initiation-defective mutant of cdc45, but not in an mcm2 or polε mutant. The results suggest that Hsk1-mediated loading of Cdc45 onto replication origins may play important roles in replication stress-induced checkpoint.