The N-terminus of the human RecQL4 helicase is a homeodomain-like DNA interaction motif

The RecQL4 helicase is involved in the maintenance of genome integrity and DNA replication. Mutations in the human RecQL4 gene cause the Rothmund–Thomson, RAPADILINO and Baller–Gerold syndromes. Mouse models and experiments in human and Xenopus have proven the N-terminal part of RecQL4 to be vital for cell growth. We have identified the first 54 amino acids of RecQL4 (RecQL4_N54) as the minimum interaction region with human TopBP1. The solution structure of RecQL4_N54 was determined by heteronuclear liquid–state nuclear magnetic resonance (NMR) spectroscopy (PDB 2KMU; backbone root-mean-square deviation 0.73 Å). Despite low-sequence homology, the well-defined structure carries an overall helical fold similar to homeodomain DNA-binding proteins but lacks their archetypical, minor groove-binding N-terminal extension. Sequence comparison indicates that this N-terminal homeodomain-like fold is a common hallmark of metazoan RecQL4 and yeast Sld2 DNA replication initiation factors. RecQL4_N54 binds DNA without noticeable sequence specificity yet with apparent preference for branched over double-stranded (ds) or single-stranded (ss) DNA. NMR chemical shift perturbation observed upon titration with Y-shaped, ssDNA and dsDNA shows a major contribution of helix α3 to DNA binding, and additional arginine side chain interactions for the ss and Y-shaped DNA.     

RecQL4: a helicase linking formation and maintenance of a replication fork

RecQ family helicases are conserved from bacteria to human. Across the species, they are known to be required for protecting genome from various genotoxic stresses. In human, five RecQ-related helicases have been identified and three of them, BLM, WRN and RecQL4, have been shown to be responsible for genetic disorders, Bloom, Werner and Rothmund-Thomson syndrome, respectively, which are characterized by cancer predisposition and premature ageing. RecQL4, the N-terminal portion of which shares similarity with Sld2 known to be required for assembly of a replication complex in yeasts, is unique in that it has been shown to be essential for the initiation phase of normal DNA replication. Recent biochemical characterization demonstrated the 3'-5' DNA helicase activity associated with RecQL4. Understanding the molecular basis for how RecQ helicases are involved in generation and maintenance of normal and stalled DNA replication forks would be crucial to elucidation of the mechanisms of replication initiation as well as to that of how the loss of these conserved helicases leads to varieties of disease phenotypes.     

Efficient Initiation of DNA Replication in Eukaryotes Requires Dpb11/TopBP1-GINS Interaction

Dpb11/Cut5/TopBP1 is evolutionarily conserved and is essential for the initiation of DNA replication in eukaryotes. The Dpb11 of the budding yeast Saccharomyces cerevisiae has four BRCT domains (BRCT1 to -4). The N-terminal pair (BRCT1 and -2) and the C-terminal pair (BRCT3 and -4) bind to cyclin-dependent kinase (CDK)-phosphorylated Sld3 and Sld2, respectively. These phosphorylation-dependent interactions trigger the initiation of DNA replication. BRCT1 and -2 and BRCT3 and -4 of Dpb11 are separated by a short stretch of ∼100 amino acids. It is unknown whether this inter-BRCT region functions in DNA replication. Here, we showed that the inter-BRCT region is a GINS interaction domain that is essential for cell growth and that mutations in this domain cause replication defects in budding yeast. We found the corresponding region in the vertebrate ortholog, TopBP1, and showed that the corresponding region also interacts with GINS and is required for efficient DNA replication. We propose that the inter-BRCT region of Dpb11 is a functionally conserved GINS interaction domain that is important for the initiation of DNA replication in eukaryotes.     

The N-terminal noncatalytic region of Xenopus RecQ4 is required for chromatin binding of DNA polymerase alpha in the initiation of DNA replication

Recruitment of DNA polymerases onto replication origins is a crucial step in the assembly of eukaryotic replication machinery. A previous study in budding yeast suggests that Dpb11 controls the recruitment of DNA polymerases alpha and epsilon onto the origins. Sld2 is an essential replication protein that interacts with Dpb11, but no metazoan homolog has yet been identified. We isolated Xenopus RecQ4 as a candidate Sld2 homolog. RecQ4 is a member of the metazoan RecQ helicase family, and its N-terminal region shows sequence similarity with Sld2. In Xenopus egg extracts, RecQ4 is essential for the initiation of DNA replication, in particular for chromatin binding of DNA polymerase alpha. An N-terminal fragment of RecQ4 devoid of the helicase domain could rescue the replication activity of RecQ4-depleted extracts, and antibody against the fragment inhibited DNA replication and chromatin binding of the polymerase. Further, N-terminal fragments of RecQ4 physically interacted with Cut5, a Xenopus homolog of Dpb11, and their ability to bind to Cut5 closely correlated with their ability to rescue the replication activity of the depleted extracts. Our data suggest that RecQ4 performs an essential role in the assembly of replication machinery through interaction with Cut5 in vertebrates.     

The N-terminal region of RECQL4 lacking the helicase domain is both essential and sufficient for the viability of vertebrate cells: Role of the N-terminal region of RECQL4 in cells

Rothmund-Thomson syndrome (RTS) is a rare genetic disorder characterized by premature aging, developmental abnormalities, and a predisposition to cancer. RTS is caused by mutations in the RECQL4 gene, which encodes one of the five human RecQ helicases. To identify the cellular functions of RECQL4, we generated a chicken DT40 cell line in which RECQL4 expression could be turned off by doxycycline (Dox). Upon exposure to Dox, cells stopped growing and underwent apoptosis. The cells could be rescued by expression of the N-terminal region of RECQL4 (amino acids 1-496), which lacks the helicase domain and has sequence similarity to yeast Sld2, which plays an essential function in the initiation of DNA replication in Saccharomyces cerevisiae. Smaller fragments of the N-terminal region of RECQL4 did not rescue the cells from lethality. RECQL4 gene knockout cells complemented with RECQL4 (1-496) showed relatively high sensitivity to DNA damaging agents that induce double strand breaks and cross-links, suggesting that the C-terminal region including the helicase domain of RECQL4 is involved in the repair of certain types of DNA lesions.     

RecQL4 is required for the association of Mcm10 and Ctf4 with replication origins in human cells

Though RecQL4 was shown to be essential for the initiation of DNA replication in mammalian cells, its role in initiation is poorly understood. Here, we show that RecQL4 is required for the origin binding of Mcm10 and Ctf4, and their physical interactions and association with replication origins are controlled by the concerted action of both CDK and DDK activities. Although RecQL4-dependent binding of Mcm10 and Ctf4 to chromatin can occur in the absence of pre-replicative complex, their association with replication origins requires the presence of the pre-replicative complex and CDK and DDK activities. Their association with replication origins and physical interactions are also targets of the DNA damage checkpoint pathways which prevent initiation of DNA replication at replication origins. Taken together, the RecQL4-dependent association of Mcm10 and Ctf4 with replication origins appears to be the first important step controlled by S phase promoting kinases and checkpoint pathways for the initiation of DNA replication in human cells.     

The N-terminal region of RECQL4 lacking the helicase domain is both essential and sufficient for the viability of vertebrate cells. Role of the N-terminal region of RECQL4 in cells

Rothmund-Thomson syndrome (RTS) is a rare genetic disorder characterized by premature aging, developmental abnormalities, and a predisposition to cancer. RTS is caused by mutations in the RECQL4 gene, which encodes one of the five human RecQ helicases. To identify the cellular functions of RECQL4, we generated a chicken DT40 cell line in which RECQL4 expression could be turned off by doxycycline (Dox). Upon exposure to Dox, cells stopped growing and underwent apoptosis. The cells could be rescued by expression of the N-terminal region of RECQL4 (amino acids 1-496), which lacks the helicase domain and has sequence similarity to yeast Sld2, which plays an essential function in the initiation of DNA replication in Saccharomyces cerevisiae. Smaller fragments of the N-terminal region of RECQL4 did not rescue the cells from lethality. RECQL4 gene knockout cells complemented with RECQL4 (1-496) showed relatively high sensitivity to DNA damaging agents that induce double strand breaks and cross-links, suggesting that the C-terminal region including the helicase domain of RECQL4 is involved in the repair of certain types of DNA lesions.     

Possible involvement of RecQL4 in the repair of double-strand DNA breaks in Xenopus egg extracts

Mutations in RecQL4 are a causative factor in Rothmund–Thomson syndrome, a human autosomal recessive disorder characterized by premature aging. To study the role of RecQL4, we employed a cell-free experimental system consisting of Xenopus egg extracts. RecQL4 loading onto chromatin was observed regardless of the presence or absence of EcoRI. However, in the absence of EcoRI, RecQL4 loading was suppressed by geminin, an inhibitor of pre-replicative complex formation, while in the presence of EcoRI, it was not affected. These results suggest that under the former condition, RecQL4-loading depended on DNA replication, while under the latter, the interaction occurred in response to double-stranded DNA breaks (DSBs) induced by EcoRI. DSB-induced RecQL4 loading depended on the function of the ataxia-telangiectasia mutated protein, DNA-dependent protein kinase (DNA-PK), and replication protein A, while there were only minor changes in DNA replication-associated RecQL4 loading upon suppression of these proteins. Furthermore, analyses using a chromatin-immunoprecipitation assay and quantification of γH2AX after induction of DSBs suggested that RecQL4 is loaded adjacent to Ku heterodimer-binding sites on damaged chromatin, and functions in the repair of DSBs.     

Initiation of DNA replication requires the RECQL4 protein mutated in Rothmund-Thomson syndrome

How the replication machinery is loaded at origins of DNA replication is poorly understood. Here, we implicate in this process the Xenopus laevis homolog (xRTS) of the RECQL4 helicase mutated in Rothmund-Thomson syndrome. xRTS, which bears homology to the yeast replication factors Sld2/DRC1, is essential for DNA replication in egg extracts. xRTS can be replaced in extracts by its human homolog, while RECQL4 depletion from mammalian cells induces proliferation failure, suggesting an evolutionarily conserved function. xRTS accumulates on chromatin during replication initiation, after prereplication-complex (pre-RC) proteins, Cut5, Sld5, or Cdc45 but before replicative polymerases. xRTS depletion suppresses the loading of RPA, the ssDNA binding protein that marks unwound origins before polymerase recruitment. However, xRTS is unaffected by xRPA depletion. Thus, xRTS functions after pre-RC formation to promote loading of replication factors at origins, a previously unrecognized activity necessary for initiation. This role connects defective replication initiation to a chromosome-fragility disorder.     

Sld3, which interacts with Cdc45 (Sld4), functions for chromosomal DNA replication in Saccharomyces cerevisiae

Cdc45, which binds to the minichromosomal maintenance (Mcm) proteins, has a pivotal role in the initiation and elongation steps of chromosomal DNA replication in eukaryotes. Here we show that throughout the cell cycle in Saccharomyces cerevisiae, Cdc45 forms a complex with a novel factor, Sld3. Consistently, Sld3 and Cdc45 associate simultaneously with replication origins in the chromatin immunoprecipitation assay: both proteins associate with early-firing origins in G(1) phase and with late-firing origins in late S phase. Moreover, the origin associations of Sld3 and Cdc45 are mutually dependent. The temperature-sensitive sld3 mutation confers a defect in DNA replication at the restrictive temperature and reduces an interaction not only between Sld3 and Cdc45, but also between Cdc45 and Mcm2. These results suggest that the Sld3-Cdc45 complex associates with replication origins through Mcm proteins. At the restrictive temperature in sld3-5 cells, replication factor A, a single-strand DNA binding protein, does not associate with origins. Therefore, the origin association of Sld3-Cdc45 complex is prerequisite for origin unwinding in the initiation of DNA replication.     

Sld7, an Sld3-associated protein required for efficient chromosomal DNA replication in budding yeast

Genetic screening of yeast for sld (synthetic lethality with dpb11) mutations has identified replication proteins, including Sld2, -3, and -5, and clarified the molecular mechanisms underlying eukaryotic chromosomal DNA replication. Here, we report a new replication protein, Sld7, identified by rescreening of sld mutations. Throughout the cell cycle, Sld7 forms a complex with Sld3, which associates with replication origins in a complex with Cdc45, binds to Dpb11 when phosphorylated by cyclin-dependent kinase, and dissociates from origins once DNA replication starts. However, Sld7 does not move with the replication fork. Sld7 binds to the nonessential N-terminal portion of Sld3 and reduces its affinity for Cdc45, a component of the replication fork. Although Sld7 is not essential for cell growth, its absence reduces the level of cellular Sld3, delays the dissociation from origins of GINS, a component of the replication fork, and slows S-phase progression. These results suggest that Sld7 is required for the proper function of Sld3 at the initiation of DNA replication.     

Treslin, DUE-B, and GEMC1 cannot complement Sld3 mutants in fission yeast

Initiation of DNA replication in eukaryotes is an evolutionarily conserved process that involves two distinct steps: the formation of prereplication complexes at replication origins in G1 and the assembly of preinitiation complexes (pre-ICs) in S phase, which leads to activation of the replication helicase. For the assembly of pre-ICs in yeast, formation of the Sld2-Dpb11-Sld3 complex is a critical event that requires phosphorylation of Sld2 and Sld3 by cyclin-dependent kinase. In mammals, RecQL4 and TopBP1 are excellent ortholog candidates for Sld2 and Dpb11, respectively. In this past year, three TopBP1-interacting proteins Treslin/Ticrr, GEMC1, and DUE-B have been identified in metazoans as possible functional orthologs of the yeast Sld3. To test this hypothesis, we carried out several complementation tests in fission yeast. The proteins were expressed at various levels in the temperature-sensitive sld3-10 mutant and in cells that lack endogenous Sld3. Our result showed that none of these metazoan proteins could rescue growth defect of the sld3 mutants. Although the result may have several interpretations, it is possible that the helicase activation in mammals has diverged in complexity during evolution from that in yeasts and may involve multiple players that interact with TopBP1.     

The Replication Initiation Protein Sld3/Treslin Orchestrates the Assembly of the Replication Fork Helicase during S Phase

The initiation of DNA replication is a highly regulated process in eukaryotic cells, and central to the process of initiation is the assembly and activation of the replication fork helicase. The replication fork helicase is comprised of CMG (Cdc45, Mcm2–7, and GINS) in eukaryotic cells, and the mechanism underlying assembly of the CMG during S phase was studied in this article. We identified a point mutation of Sld3 that is specifically defective for Mcm3 and Mcm5 interaction (sld3-m10), and also identified a point mutation of Sld3 that is specifically defective for single-stranded DNA (ssDNA) interaction (sld3-m9). Expression of wild-type levels of sld3-m9 resulted in a severe DNA replication defect with no recruitment of GINS to Mcm2–7, whereas expression of wild-type levels of sld3-m10 resulted in a severe replication defect with no Cdc45 recruitment to Mcm2–7. We propose a model for Sld3-mediated control of replication initiation, wherein Sld3 manages the proper assembly of the CMG during S phase. We also find that the biochemical functions identified for Sld3 are conserved in human Treslin, suggesting that Treslin orchestrates assembly of the CMG in human cells.     

Human Origin Recognition Complex Binds Preferentially to G-quadruplex-preferable RNA and Single-stranded DNA

Origin recognition complex (ORC), consisting of six subunits ORC1–6, is known to bind to replication origins and function in the initiation of DNA replication in eukaryotic cells. In contrast to the fact that Saccharomyces cerevisiae ORC recognizes the replication origin in a sequence-specific manner, metazoan ORC has not exhibited strict sequence-specificity for DNA binding. Here we report that human ORC binds preferentially to G-quadruplex (G4)-preferable G-rich RNA or single-stranded DNA (ssDNA). We mapped the G-rich RNA-binding domain in the ORC1 subunit, in a region adjacent to its ATPase domain. This domain itself has an ability to preferentially recognize G4-preferable sequences of ssDNA. Furthermore, we found, by structure modeling, that the G-rich RNA-binding domain is similar to the N-terminal portion of AdoMet_MTase domain of mammalian DNA methyltransferase 1. Therefore, in contrast with the binding to double-stranded DNA, human ORC has an apparent sequence preference with respect to its RNA/ssDNA binding. Interestingly, this specificity coincides with the common signature present in most of the human replication origins. We expect that our findings provide new insights into the regulations of function and chromatin binding of metazoan ORCs.     

Phosphopeptide binding by Sld3 links Dbf4‐dependent kinase to MCM replicative helicase activation

The initiation of eukaryotic DNA replication requires the assembly of active CMG (Cdc45‐MCM‐GINS) helicases at replication origins by a set of conserved and essential firing factors. This process is controlled during the cell cycle by cyclin‐dependent kinase (CDK) and Dbf4‐dependent kinase (DDK), and in response to DNA damage by the checkpoint kinase Rad53/Chk1. Here we show that Sld3, previously shown to be an essential CDK and Rad53 substrate, is recruited to the inactive MCM double hexamer in a DDK‐dependent manner. Sld3 binds specifically to DDK‐phosphorylated peptides from two MCM subunits (Mcm4, 6) and then recruits Cdc45. MCM mutants that cannot bind Sld3 or Sld3 mutants that cannot bind phospho‐MCM or Cdc45 do not support replication. Moreover, phosphomimicking mutants in Mcm4 and Mcm6 bind Sld3 without DDK and facilitate DDK‐independent replication. Thus, Sld3 is an essential “reader” of DDK phosphorylation, integrating signals from three distinct protein kinase pathways to coordinate DNA replication during S phase.     

Multiple regulatory mechanisms to inhibit untimely initiation of DNA replication are important for stable genome maintenance

Genomic instability is a hallmark of human cancer cells. To prevent genomic instability, chromosomal DNA is faithfully duplicated in every cell division cycle, and eukaryotic cells have complex regulatory mechanisms to achieve this goal. Here, we show that untimely activation of replication origins during the G1 phase is genotoxic and induces genomic instability in the budding yeast Saccharomyces cerevisiae. Our data indicate that cells preserve a low level of the initiation factor Sld2 to prevent untimely initiation during the normal cell cycle in addition to controlling the phosphorylation of Sld2 and Sld3 by cyclin-dependent kinase. Although untimely activation of origin is inhibited on multiple levels, we show that deregulation of a single pathway can cause genomic instability, such as gross chromosome rearrangements (GCRs). Furthermore, simultaneous deregulation of multiple pathways causes an even more severe phenotype. These findings highlight the importance of having multiple inhibitory mechanisms to prevent the untimely initiation of chromosome replication to preserve stable genome maintenance over generations in eukaryotes.     

Sld2 binds to origin single-stranded DNA and stimulates DNA annealing

Sld2 is essential for the initiation of DNA replication, but the mechanism underlying its role in replication is not fully understood. The S-phase cyclin dependent kinase (S-CDK) triggers the association of Sld2 with Dpb11, and a phosphomimetic mutation of Sld2, Sld2T84D, functionally mimics the S-CDK phosphorylated state of Sld2. We report that Sld2T84D binds directly to the single-stranded (ss) DNA of two different origins of replication, and S-CDK phosphorylation of Sld2 stimulates the binding of Sld2 to origin ssDNA. Sld2T84D binds to a thymine-rich ssDNA region of the origin ARS1, and substitution of ARS1 thymines with adenines completely disrupts binding of Sld2T84D. Sld2T84D enhances the ability of origin ssDNA to pulldown Dpb11, and Sld2 binding to origin ssDNA may be important to allow Sld2 and Dpb11 to associate with origin DNA. We also report that Sld2T84D anneals ssDNA of an origin sequence. Dpb11 anneals ssDNA to low levels, and the addition of Sld2T84D with Dpb11 results in higher annealing activity than that of either protein alone. Sld2-stimulated annealing may be important for maintaining genome stability during the initiation of DNA replication.     

Dual DNA unwinding activities of the Rothmund–Thomson syndrome protein,RECQ4

Human RECQ helicases have been linked to distinct clinical diseases with increased cancer rates and premature ageing. All RECQ proteins, except RECQ4, have been shown to be functional helicases. Mutations in RECQ4 lead to Rothmund–Thomson syndrome (RTS), and mouse models reveal that the conserved helicase motifs are required for avoidance of RTS. Furthermore, the amino (N) terminus of RECQ4 shares homology with yeast DNA replication initiation factor, Sld2, and is vital for embryonic development. Here, in contrast to previous reports, we show that RECQ4 exhibits DNA helicase activity. Importantly, two distinct regions of the protein, the conserved helicase motifs and the Sld2‐like N‐terminal domain, each independently promote ATP‐dependent DNA unwinding. Taken together, our data provide the first biochemical clues underlying the molecular function of RECQ4 in DNA replication and genome maintenance.     

A positive involvement of RecQL4 in UV-induced S-phase arrest

RecQL4 belongs to a family of conserved RECQ helicases that are important in maintaining chromosomal integrity. Human patients lacking RecQL4 showed extreme sensitivity to UV and oxidation damage, suggesting that RecQL4 is involved in the damage signaling and/or repair. Here we show that human mutant cells lacking RecQL4 were defective in UV-induced S-phase arrest, whereas cells defective in bloom syndrome protein (BLM), another member of RecQ family exhibited a normal S-phase arrest following UV irradiation. In keeping with this, a targeted inhibition of RecQL4 expression in human 293 cells showed a defect in inducing S-phase (replication) arrest following UV treatment. Human mutant cells lacking RecQL4 protein were also defective in inducing S-phase arrest following hydroxyurea treatment. Together, our results suggest that RecQL4 may have a unique role in replication fork arrest, which may not be shared with other members of RecQ family such as BLM.     

Origin association of Sld3, Sld7, and Cdc45 proteins is a key step for determination of origin-firing timing

Background

Chromosomal DNA replication in eukaryotes initiates from multiple origins of replication, and because of this multiplicity, activation of replication origins is likely to be highly coordinated; origins fire at characteristic times, with some origins firing on average earlier (early-firing origins) and others later (late-firing origins) in the S phase of the budding yeast cell cycle. However, the molecular basis for such temporal regulation is poorly understood.

Results

We show that origin association of the low-abundance replication proteins Sld3, Sld7, and Cdc45 is the key to determining the temporal order of origin firing. These proteins form a complex and associate with the early-firing origins in G1 phase in a manner that depends on Dbf4-dependent kinase (DDK), which is essential for the initiation of DNA replication. An increased dosage of Sld3, Sld7, and Cdc45 allows the late-firing origins to fire earlier in S phase. Additionally, an increased dosage of DDK also allows the late-firing origins to fire earlier.

Conclusions

The DDK-dependent limited association between origins and Sld3-Sld7-Cdc45 is a key step for determining the timing of origin firing.