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TdIF1 was originally identified as a protein that directly binds to terminal deoxynucleotidyltransferase, TdT. Through in vitro selection assays (SELEX), we recently showed that TdIF1 recognizes both AT-tract and a specific DNA sequence motif, 5'-TGCATG-3', and can up-regulate the expression of RAB20 through the latter motif. However, whether TdIF1 binds to these sequences in the cells has not been clear and its other target genes remain to be identified. Here, we determined in vivo TdIF1-binding sequences (TdIF1-invivoBMs) on the human chromosomes through ChIP-seq analyses. The result showed a 160-base pair cassette containing 'AT-tract~palindrome (inverted repeat)~AT-tract' as a likely target sequence of TdIF1. Interestingly, the core sequence of the palindrome in the TdIF1-invivoBMs shares significant similarity to the above 5'-TGCATG-3' motif determined by SELEX in vitro. Furthermore, spacer sequences between AT-tract and the palindrome contain many potential transcription factor binding sites. In luciferase assays, TdIF1 can up-regulate transcription activity of the promoters containing the TdIF1-invivoBM, and this effect is mainly through the palindrome. Clusters of this motif were found in the potential target genes. Gene ontology analysis and RT-qPCR showed the enrichment of some candidate targets of TdIF1 among the genes involved in the regulation of ossification. Potential modes of transcription activation by TdIF1 are discussed.

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.

Purification of recombinant proteins of a large size often poses problems of instability or low expression in bacterial or insect cells. Here, we established a method for a high-level expression of large-sized recombinant proteins in mammalian cells and subsequent purification of the full-length proteins. We applied this method to express human Claspin and Tim–Tipin complex, which play important roles in replication checkpoint responses as fork-stabilizing factors, and successfully purified them in functional forms in amount sufficient for enzymatic characterization. Purified Claspin behaves as a monomer and binds preferentially to fork-like DNA. Over-expression of tagged Claspin in mammalian cells facilitated the detection of its interacting factors. Claspin interacts with many factors involved in checkpoint regulation and replication fork machinery, including ATR, ATM, Chk1, Tim, MCM4, MCM10, Cdc45, DNA polymerases α, δ, e and Cdc7 kinase. We will discuss the potential implication of these findings in architecture of replication fork. We will also discuss the advantage of this system for purification and characterization of those proteins that are large and have been difficult to deal with.

In bacteria, PriA protein, a conserved DEXH-type DNA helicase, plays a central role in replication restart at stalled replication forks. Its unique DNA-binding property allows it to recognize and stabilize stalled forks and the structures derived from them. Cells must cope with fork stalls caused by various replication stresses to complete replication of the entire genome. Failure of the stalled fork stabilization process and eventual restart could lead to various forms of genomic instability. The low viability of priA null cells indicates a frequent occurrence of fork stall during normal growth that needs to be properly processed. PriA specifically recognizes the 3'-terminus of the nascent leading strand or the invading strand in a displacement (D)-loop by the three-prime terminus binding pocket (TT-pocket) present in its unique DNA binding domain. Elucidation of the structural basis for recognition of arrested forks by PriA should provide useful insight into how stalled forks are recognized in eukaryotes.

The checkpoint clamp Rad9-Hus1-Rad1 (9-1-1) is loaded by the Rad17–RFC complex onto chromatin after DNA damage and plays a key role in the ATR-dependent checkpoint activation. Here, we demonstrate that in vitro casein kinase 2 (CK2) specifically interacts with human 9-1-1 and phosphorylates serines 341 and 387 (Ser-341 and Ser-387) in the C-terminal tail of Rad9. Interestingly, phosphorylated Ser-387 has previously been reported to be required for interacting with a checkpoint mediator TopBP1. Indeed, 9-1-1 purified from Escherichia coli and phosphorylated in vitro by CK2 physically interacts with TopBP1. Further analyses showed that phosphorylation at both serine residues occurs in vivo and is required for the efficient interaction with TopBP1 in vitro. Furthermore, when over-expressed in HeLa cells, a mutant Rad9 harboring phospho-deficient substitutions at both Ser-341 and Ser-387 residues causes hypersensitivity to UV and methyl methane sulfonate (MMS). Our observations suggest that CK2 plays a crucial role in the ATR-dependent checkpoint pathway through its ability to phosphorylate Ser-341 and Ser-387 of the Rad9 subunit of the 9-1-1 complex.

Cohesin-mediated sister chromatid cohesion is established during the S-phase, and recent studies demonstrate that a cohesin protein ring concatenates sister DNA molecules. However, little is known about how DNA replication is linked to the establishment of sister chromatid cohesion. Here, we used Xenopus egg extracts to show that AND-1 and Tim1-Tipin, homologues of Saccharomyces cerevisiae Ctf4 and Tof1-Csm3, respectively, are associated with the replisome and are required for proper establishment of the cohesion observed in the M-phase extracts. Immunodepletion of both AND-1 and Tim1-Tipin from the extracts leads to aberrant sister chromatid cohesion, which is similarly induced by the depletion of cohesin. These results demonstrate that AND-1 and Tim1-Tipin are key factors linking DNA replication and establishment of sister chromatid cohesion. On the basis of the physical interactions between AND-1 and DNA polymerases, we discuss a model to describe how replisome progression complex establishes sister chromatid cohesion.

ASK (activator of S phase kinase) is an activation subunit for mammalian Cdc7 kinase. We have generated mutant ES cell lines in which ASK can be conditionally inactivated. Upon loss of the ASK genes, the mutant ES cells rapidly cease cell growth. In keeping with its expected roles in activation of the essential S phase kinase, DNA synthesis is arrested and significant cell death is eventually induced in ASK-deficient cells, demonstrating essential roles of ASK for viability of ES cells. Using these mutant cells, we have set up a system where ASK molecules can be functionally dissected. In keeping with previous results from yeasts, conserved motif-M and motif-C were shown to be essential for in vivo functions of ASK, whereas a long C-terminal tail, found only in ASK-related molecules in higher eukaryotes, is not required. Unexpectedly, the motif-N, related to the BRCT motif and dispensable for viability in yeasts, is essential for the viability of ES cells. Further characterization reveals that motif-N is required for the maximum phosphorylation of MCM in cells, whereas the autophosphorylation activity of Cdc7 is not significantly affected by its loss. These results may suggest that motif-N of ASK may facilitate recruitment of substrates for Cdc7 kinase.

The presence of multiple clusters of runs of asymmetric adenine or thymine is a feature commonly found in eukaryotic replication origins. Here we report that the helicase and ATPase activities of the mammalian Mcm4/6/7 complex are activated specifically by thymine stretches. The Mcm helicase is specifically activated by a synthetic bubble structure which mimics an activated replication origin, as well as by a Y-fork structure, provided that a single-stranded DNA region of sufficient length is present in the unwound segment or 3′ tail, respectively, and that it carries clusters of thymines. Sequences derived from the human lamin B2 origin can serve as a potent activator for the Mcm helicase, and substitution of its thymine clusters with guanine leads to loss of this activation. At the fork, Mcm displays marked processivity, expected for a replicative helicase. These findings lead us to propose that selective activation by stretches of thymine sequences of a fraction of Mcm helicases loaded onto chromatin may be the determinant for selection of initiation sites on mammalian genomes.

Cdc7 kinase is essential for initiation of DNA replication. Cdc7(-/-) mouse embryonic stem (ES) cells are non-viable but their growth can be rescued by an ectopically expressed transgene (Cdc7(-/-)tg). Here we report that, despite the normal growth capability of Cdc7(-/-)tg ES cells, the mice with the identical genetic background exhibit growth retardation. Concomi tantly, Cdc7(-/-)tg embryonic fibroblasts (MEFs) display delayed S phase entry and slow S phase progression. Furthermore, spermatogenesis of Cdc7(-/-)tg mice is disrupted prior to pachytene stage of meiotic prophase I. The impairment in spermatogenesis correlates with the extremely low level of Cdc7 protein in testes, and is rescued by introducing an additional allele of transgene, which results in increase of Cdc7 expression. The increased level of Cdc7 also recovers the growth of Cdc7(-/-)tg MEFs and mice, indicating that the developmental abnormalities of Cdc7(-/-)tg mice are due to insufficiency of Cdc7 protein. Our results indicate the requirement of a critical level of a cell-cycle regulator for mouse development and provide genetic evidence that Cdc7 plays essential roles in meiotic processes in mammals.

Background: During the course of DNA replication, regulation of cellular localization and chromatin binding of involved factors plays critical roles. Cdc7 kinase is required for DNA replication and its kinase activity is cell cycle-regulated by its activation subunit Dbf4/ASK. In mammals, it is not known at which time point during the cell cycle Cdc7 and Dbf4/ASK proteins are imported into nuclei and loaded on to chromatin. Results: We have constructed a series of truncation and deletion derivatives of ASK and expressed them as fusion proteins with GFP in mammalian cells. Both Dbf4-motif-M and -C conserved in Dbf4/ASK protein family are required for huCdc7 kinase activation. Two stretches of amino acid sequences, NLS1 (P346KKKRIK) and NLS2 (K201RVGSGAQKTRTGRLKK), are important for ASK nuclear localization. In stable transformants expressing GFP-fused full-length ASK under the tetracycline inducible promoter, GFP-ASK protein accumulates in nuclei at the telophase, but its binding to chromatin does not reach a maximum until late G1, whereas huCdc7 is imported into nuclei and binds to chromatin at early G1. An important substrate of Cdc7-ASK at the G1/S transition is likely to be MCM. Indeed, over-expression of both huCdc7 and ASK results in the elevated phosphorylation of endogenous MCM2 protein, as manifested by appearance of the mobility-shifted form on SDS-PAGE, but does not cause any significant effects on cell cycle progression. Conclusions: Nuclear localization and chromatin binding of endogenous huCdc7 and GFP-ASK expressed during the post-mitotic phase are independently regulated. Although GFP-ASK is presumably imported into nuclei through its two nuclear localization signals at telophase, it may require additional signals for chromatin binding, the level of which increases at late G1 phase.Summary figure Figure Summary figure . Cell cycle regulation of cellular localization and chromatin binding of Cdc7 and ASK proteins. See text for details. Download figure to PowerPoint

Backgrounds: PriA protein, a DEXH-type helicase with C2C2 zinc-finger motifs, plays essential roles in RecA-dependent modes of Escherichia coli chromosomal DNA replication, namely inducible and constitutive stable DNA replication (iSDR and cSDR respectively, which may be initiated from a D-loop or R-loop structure), and in repair of double-stranded DNA breaks generated by various genotoxic agents or spontaneously during the course of DNA replication. However, the roles of ATPase/DNA helicase activities in functions of PriA are not well understood. Results: We have generated and characterized mutants of PriA protein carrying amino acid substitutions in its conserved ATPase/DNA helicase motifs, namely the Walker A, B and QXXGRXGR motifs. All these mutants were deficient in ATP hydrolysis and DNA helicase activities, but showed wild-type levels of D-loop DNA binding, except for the Walker B mutant which showed reduced DNA binding activity, suggesting that the helicase motifs are not directly involved in the DNA binding activity of PriA protein. They also rescued the low viability and UV-sensitivity of priA null cells. However, they did not rescue iSDR or cSDR—alternative modes of chromosomal DNA replication of the E. coli genome dependent on recombination functions—to the full extent. Conclusions: ATPase/DNA helicase activities of PriA protein are required for full-level DNA synthesis in recombination-dependent modes of DNA replication in E. coli.

Cdc7‐related kinases play essential roles in the initiation of yeast DNA replication. We show that mice lacking murine homologs of Cdc7 ( muCdc7 ) genes die between E3.5 and E6.5. We have established a mutant embryonic stem (ES) cell line lacking the muCdc7 genes in the presence of a loxP ‐flanked transgene expressing muCdc7 cDNA. Upon removal of the transgene by Cre recombinase, mutant ES cells cease DNA synthesis, arresting growth with S‐phase DNA content, and generate nuclear Rad51 foci, followed by cell death with concomitant increase in p53 protein levels. Inhibition of p53 leads to partial rescue of muCdc7 −/− ES cells from cell death. muCdc7 −/− p53 −/− embryos survive up to E8.5, and their blastocysts generate inner cell mass of a significant size in vitro , whereas those of the muCdc7 −/− p53 +/− embryos undergoes complete degeneration. These results demonstrate that, in contrast to cell cycle arrest at the G 1 /S boundary observed in yeasts, loss of Cdc7 in ES cells results in rapid cessation of DNA synthesis within S phase, triggering checkpoint responses leading to recombinational repair and p53‐dependent cell death.

DNA replication results from the action of a staged set of highly regulated processes. Among the stages of DNA replication, initiation is the key point at which all the G1 regulatory signals culminate. Cdc7 kinase is the critical regulator for the ultimate firing of the origins of initiation. Cdc7, originally identified in budding yeast and later in higher eukaryotes, forms a complex with a Dbf4-related regulatory subunit to generate an active kinase. Genetic evidence in mammals demonstrates essential roles for Cdc7 in mammalian DNA replication. Mini-chromosome maintenance protein (MCM) is the major physiological target of Cdc7. Genetic studies in yeasts indicate additional roles of Cdc7 in meiosis, checkpoint responses, maintenance of chromosome structures, and repair. The interplay between Cdc7 and Cdk, another kinase essential for the S phase, is also discussed.

South-Western screening of a glutathione-S-transferase fusion protein library constructed from the yeast Saccharomyces cerevisiae genomic DNA lead to isolation of core T-rich-strand-binding protein (CTBP) clones that bound to single-stranded DNA containing the T-rich-strand of the 11-bp core sequence of autonomously replicating sequences. One of these clones, CTBP1, contains a portion of previously described RBP1 which is an RNA-binding and single-stranded DNA-binding protein of S. cerevisiae. GST-CTBP1 as well as the full-length fusion protein with RBP1 (GST-RBP1) bind exclusively to the T-rich strand of the core sequence with an apparent dissociation constant of 5 nM, but not to the A-rich strand or double strand of the same sequence. Mutations within the core which reduce the number of T or C residues decrease the affinity of this protein. In keeping with this, binding of GST-CTBP1 to the core sequence is efficiently completed by poly(dT), poly(dT-dC) or poly(dC), but not by poly(dA) or poly(dG) to significant extents. Among polyribonucleic acids, GST-CTBP1 binds to poly(U) and poly(I) with greatest affinity, whereas GST-RBP1 binds to RNA in a rather non-specific manner. In no cases was affinity for RNA greater than that for DNA. Our results indicate that CTBP1/RBP1 is a polydeoxypyrimidine-binding protein of S. cerevisiae. CTBP1 contains two sets of an RNA-recognition motif (RRM) and a glutamine stretch. The binding affinity of the N-terminal or C-terminal set containing one RRM and one glutamine stretch is nearly two orders of magnitude lower than that of the wild-type CTBP1 containing both sets. The isolated N-terminal or C-terminal RRM alone (RRM1 and RRM2, respectively) is sufficient for binding nucleic acids with the binding specificity similar to that of the wild-type RRM, although the binding affinity of the isolated RRM2 is nearly two orders of magnitude lower than that of RRM1. Our results indicate that the two RRMs present in CTBP1/RBP1 have differential binding affinities and that the high affinity of RRM for polydeoxypyrimidine results from synergy between two lower-affinity RRMs.

The ABC primosome is assembled from DnaA, DnaB and DnaC proteins at a stem-and-loop structure containing a dnaA box within its stem (A site), and catalyses primer RNA synthesis for DNA chain elongation. The DnaA protein can bind to the A site and the A-site-DnaA-protein complex can be isolated by gel-filtration chromatography in the absence of nucleotides. Mutations within the dnaA box completely abolish the binding of DnaA protein. Point mutations within the stem region outside the dnaA box also severely reduce the affinity of DnaA protein for the A site. These results indicate that not only the dnaA box but also other nucleotides and/or secondary structure features of the stem are important for proper recognition of the A site by DnaA protein. The preprimosome, which is able to synthesize RNA primers upon addition of primase, can be isolated by gel-filtration chromatography in the presence of ATP or adenosine 5'-[gamma-thio]triphosphate, a non-hydrolyzable analogue of ATP. The preprimosome can translocate along Escherichia coli single-stranded-DNA-binding protein-coated single-stranded DNA, utilizing the energy released by hydrolysis of ATP, as indicated by its helicase activity. dATP, as well as dCTP, can support the helicase activity of the preprimosome to some extent, while they are inert in helicase assays with DnaB protein in the absence of E. coli single-stranded DNA-binding protein. In keeping with this result, the isolated preprimosome, which appears to contain DnaA and DnaB proteins, is capable of hydrolyzing dATP as well as ATP and GTP. In a reconstituted replication assay, addition of excess dATP restores replication activities which have been inhibited by addition of adenosine 5'-[gamma-thio]triphosphate. The ability of dATP to support helicase and replicative activities of the ABC primosome indicates that the formation of the complex somehow modulates the structures of its component(s) so that they can utilize otherwise inert nucleotides. On the basis of these results, a scheme for the assembly of the ABC primosome at the A site is presented.

Under certain conditions, Escherichia coli cells exhibit either of two altered modes of chromosomal DNA replication. These are inducible stable DNA replication (iSDR), seen in SOS-induced cells, and constitutive stable DNA replication (cSDR), seen in rnhA mutants. Both iSDR and cSDR can continue to occur in the absence of protein synthesis. They are dependent on RecA protein, but do not require DnaA protein or the oriC site. Here we report the requirement for PriA, a protein essential for assembly of the phi X174-type primosome, for both iSDR and cSDR. In priA1(Null)::kan mutant cells, iSDR is not observed after induction by thymine starvation. Replication from one of the origins (oriM1) specific to iSDR is greatly reduced by the priA1::kan mutation. cSDR in rnhA224 mutant cells deficient in RNase HI is also completely abolished by the same priA mutation. In both cases, SDR is restored by introduction of a plasmid carrying a wild-type priA gene. Furthermore, the viability of an rnhA::cat dnaA46 strain is lost at 42 degrees C upon inactivation of the priA gene, indicating the lethal effect of priA inactivation on those cells whose viability depends on cSDR. These results demonstrate that a function of PriA protein is essential for iSDR and cSDR and suggest the involvement of the PriA-dependent phi X174-type primosome in these DnaA/oriC-independent pathways of chromosome replication. Whereas ColE1-type plasmids, known to be independent of DnaA, absolutely require PriA function for replication, DnaA-dependent plasmid replicons such as pSC101, F, R6K, Rts1 and RK2 are able to transform and to be maintained in the priA1::kan strain.(ABSTRACT TRUNCATED AT 250 WORDS)

How can an antiparallel DNA (deoxyribonucleic acid) strand be duplicated by a DNA polymerase that synthesises DNA in only one direction? This paradox of DNA synthesis on the lagging strand was dissolved by discovery of Okazaki fragments. The major components of the bacterial replication fork include replicative helicase, primase and DNA polymerase. The loading of replicative helicase, DnaB, is the most critical step for assembly of a primosome, a protein complex responsible for duplex unwinding and primer RNA (ribonucleic acid) synthesis at the replication fork. DNA polymerase may be an asymmetric dimer, each of which may concurrently synthesise leading or lagging strand. Several different modes of primosome assembly have been identified in bacteria. At oriC (origin of chromosome), DnaA-dependent primosome is assembled for initiation of a round of DNA replication, whereas PriA-dependent primosome is assembled at stalled replication forks to facilitate replication restart. Key Concepts: Initiation of DNA replication: DNA replication is initiated by the initiator protein, which specifically recognises and binds to the origin sequence and recruits other primosome components including a DNA helicase. Leading and lagging strands: Leading strand is the one in which the direction of DNA chain elongation and overall fork movement is the same and lagging strand is the one in which they are opposite. Replicative helicase: An enzyme which catalyses continuous unwinding of the parental duplex DNA at the replication fork. Replication fork: The site of DNA replication where two replicating single-stranded DNA separates. Primer RNA: A short stretch of RNA, the 3′-terminus of which is utilised by DNA polymerases for DNA elongation. Primosome: A name given to the protein complex capable of duplex DNA unwinding and primer RNA synthesis at the replication fork. Stalled replication fork: A replication fork the movement of which is blocked by internal and external ‘replication stress’ including DNA damages and depletion of nucleotide precursors. Replication restart: The process of reassembly of primosome at a stalled replication fork to resume DNA chain elongation. Keywords: replication fork; Okazaki fragment; DNA polymerases; primer RNA; replication restart