The N Terminus of the Retinoblastoma Protein Inhibits DNA Replication via a Bipartite Mechanism Disrupted in Partially Penetrant Retinoblastomas

The N-terminal domain of the retinoblastoma (Rb) tumor suppressor protein (RbN) harbors in-frame exon deletions in partially penetrant hereditary retinoblastomas and is known to impair cell growth and tumorigenesis. However, how such RbN deletions contribute to Rb tumor- and growth-suppressive functions is unknown. Here we establish that RbN directly inhibits DNA replication initiation and elongation using a bipartite mechanism involving N-terminal exons lost in cancer. Specifically, Rb exon 7 is necessary and sufficient to target and inhibit the replicative CMG helicase, resulting in the accumulation of inactive CMGs on chromatin. An independent N-terminal loop domain, which forms a projection, specifically blocks DNA polymerase α (Pol-α) and Ctf4 recruitment without affecting DNA polymerases ε and δ or the CMG helicase. Individual disruption of exon 7 or the projection in RbN or Rb, as occurs in inherited cancers, partially impairs the ability of Rb/RbN to inhibit DNA replication and block G₁-to-S cell cycle transit. However, their combined loss abolishes these functions of Rb. Thus, Rb growth-suppressive functions include its ability to block replicative complexes via bipartite, independent, and additive N-terminal domains. The partial loss of replication, CMG, or Pol-α control provides a potential molecular explanation for how N-terminal Rb loss-of-function deletions contribute to the etiology of partially penetrant retinoblastomas.     

The domain structure of Helicobacter pylori DnaB helicase: the N-terminal domain can be dispensable for helicase activity whereas the extreme C-terminal region is essential for its function

Hexameric DnaB type replicative helicases are essential for DNA strand unwinding along with the direction of replication fork movement. These helicases in general contain an amino terminal domain and a carboxy terminal domain separated by a linker region. Due to the lack of crystal structure of a full-length DnaB like helicase, the domain structure and function of these types of helicases are not clear. We have reported recently that Helicobacter pylori DnaB helicase is a replicative helicase in vitro and it can bypass Escherichia coli DnaC activity in vivo. Using biochemical, biophysical and genetic complementation assays, here we show that though the N-terminal region of HpDnaB is required for conformational changes between C6 and C3 rotational symmetry, it is not essential for in vitro helicase activity and in vivo function of the protein. Instead, an extreme carboxy terminal region and an adjacent unique 34 amino acid insertion region were found to be essential for HpDnaB activity suggesting that these regions are important for proper folding and oligomerization of this protein. These results confer great potential in understanding the domain structures of DnaB type helicases and their related function.     

Interaction of Rep and DnaB on DNA

Genome duplication requires not only unwinding of the template but also the displacement of proteins bound to the template, a function performed by replicative helicases located at the fork. However, accessory helicases are also needed since the replicative helicase stalls occasionally at nucleoprotein complexes. In Escherichia coli, the primary and accessory helicases DnaB and Rep translocate along the lagging and leading strand templates, respectively, interact physically and also display cooperativity in the unwinding of model forked DNA substrates. We demonstrate here that this cooperativity is displayed only by Rep and not by other tested helicases. ssDNA must be exposed on the leading strand template to elicit this cooperativity, indicating that forks blocked at protein-DNA complexes contain ssDNA ahead of the leading strand polymerase. However, stable Rep-DnaB complexes can form on linear as well as branched DNA, indicating that Rep has the capacity to interact with ssDNA on either the leading or the lagging strand template at forks. Inhibition of Rep binding to the lagging strand template by competition with SSB might therefore be critical in targeting accessory helicases to the leading strand template, indicating an important role for replisome architecture in promoting accessory helicase function at blocked replisomes.     

Structure and evolutionary origins of the CMG complex

The CMG (Cdc45–MCM–GINS) complex is the eukaryotic replicative helicase, the enzyme that unwinds double-stranded DNA at replication forks. All three components of the CMG complex are essential for its function, but only in the case of MCM, the molecular motor that harnesses the energy of ATP hydrolysis to catalyse strand separation, is that function clear. Here, we review current knowledge of the three-dimensional structure of the CMG complex and its components and highlight recent advances in our understanding of its evolutionary origins.     

DNA Structure Specificity Conferred on a Replicative Helicase by Its Loader

Prokaryotic and eukaryotic replicative helicases can translocate along single-stranded and double-stranded DNA, with the central cavity of these multimeric ring helicases being able to accommodate both forms of DNA. Translocation by such helicases along single-stranded DNA results in the unwinding of forked DNA by steric exclusion and appears critical in unwinding of parental strands at the replication fork, whereas translocation over double-stranded DNA has no well-defined role. We have found that the accessory factor, DnaC, that promotes loading of the Escherichia coli replicative helicase DnaB onto single-stranded DNA may also act to confer DNA structure specificity on DnaB helicase. When present in excess, DnaC inhibits DnaB translocation over double-stranded DNA but not over single-stranded DNA. Inhibition of DnaB translocation over double-stranded DNA requires the ATP-bound form of DnaC, and this inhibition is relieved during translocation over single-stranded DNA indicating that stimulation of DnaC ATPase is responsible for this DNA structure specificity. These findings demonstrate that DnaC may provide the DNA structure specificity lacking in DnaB, limiting DnaB translocation to bona fide replication forks. The ability of other replicative helicases to translocate along single-stranded and double-stranded DNA raises the possibility that analogous regulatory mechanisms exist in other organisms.     

Interaction of heliquinomycin with single-stranded DNA inhibits MCM4/6/7 helicase

The antibiotic heliquinomycin inhibited cellular DNA replication at IC(50) of 2.5 μM without affecting level of chromatin-bound MCM4 and without activating the DNA replication stress checkpoint system, suggesting that heliquinomycin perturbs DNA replication mainly by inhibiting the activity of replicative DNA helicase that unwinds DNA duplex at replication forks. Among the DNA helicases involved in DNA replication, DNA helicase B was inhibited by heliquinomycin at IC(50) of 4.3 μM and RECQL4 helicase at IC(50) of 14 μM; these values are higher than that of MCM4/6/7 helicase (2.5 μM). These results suggest that heliquinomycin mainly targets actions of the replicative DNA helicases. Gel-retardation experiment indicates that heliquinomycin binds to single-stranded DNA. The single-stranded DNA-binding ability of MCM4/6/7 was affected in the presence of heliquinomycin. The data suggest that heliquinomycin inhibits the DNA helicase activity of MCM4/6/7 complex by stabilizing its interaction with single-stranded DNA.     

T7 DNA helicase: a molecular motor that processively and unidirectionally translocates along single-stranded DNA

DNA helicases are molecular motors that use the energy from NTP hydrolysis to drive the process of duplex DNA strand separation. Here, we measure the translocation and energy coupling efficiency of a replicative DNA helicase from bacteriophage T7 that is a member of a class of helicases that assembles into ring-shaped hexamers. Presteady state kinetics of DNA-stimulated dTTP hydrolysis activity of T7 helicase were measured using a real time assay as a function of ssDNA length, which provided evidence for unidirectional translocation of T7 helicase along ssDNA. Global fitting of the kinetic data provided an average translocation rate of 132 bases per second per hexamer at 18 degrees C. While translocating along ssDNA, T7 helicase hydrolyzes dTTP at a rate of 49 dTTP per second per hexamer, which indicates that the energy from hydrolysis of one dTTP drives unidirectional movement of T7 helicase along two to three bases of ssDNA. One of the features that distinguishes this ring helicase is its processivity, which was determined to be 0.99996, which indicated that T7 helicase travels on an average about 75kb of ssDNA before dissociating. We propose that the ability of T7 helicase to translocate unidirectionally along ssDNA in an efficient manner plays a crucial role in DNA unwinding.     

Translocation and Stability of Replicative DNA Helicases upon Encountering DNA-Protein Cross-links

DNA-protein cross-links (DPCs) are formed when cells are exposed to various DNA-damaging agents. Because DPCs are extremely large, steric hindrance conferred by DPCs is likely to affect many aspects of DNA transactions. In DNA replication, DPCs are first encountered by the replicative helicase that moves at the head of the replisome. However, little is known about how replicative helicases respond to covalently immobilized protein roadblocks. In the present study we elucidated the effect of DPCs on the DNA unwinding reaction of hexameric replicative helicases in vitro using defined DPC substrates. DPCs on the translocating strand but not on the nontranslocating strand impeded the progression of the helicases including the phage T7 gene 4 protein, simian virus 40 large T antigen, Escherichia coli DnaB protein, and human minichromosome maintenance Mcm467 subcomplex. The impediment varied with the size of the cross-linked proteins, with a threshold size for clearance of 5.0–14.1 kDa. These results indicate that the central channel of the dynamically translocating hexameric ring helicases can accommodate only small proteins and that all of the helicases tested use the steric exclusion mechanism to unwind duplex DNA. These results further suggest that DPCs on the translocating and nontranslocating strands constitute helicase and polymerase blocks, respectively. The helicases stalled by DPC had limited stability and dissociated from DNA with a half-life of 15–36 min. The implications of the results are discussed in relation to the distinct stabilities of replisomes that encounter tight but reversible DNA-protein complexes and irreversible DPC roadblocks.     

DNA repair and replication fork helicases are differentially affected by alkyl phosphotriester lesion

DNA helicases are directly responsible for catalytically unwinding duplex DNA in an ATP-dependent and directionally specific manner and play essential roles in cellular nucleic acid metabolism. It has been conventionally thought that DNA helicases are inhibited by bulky covalent DNA adducts in a strand-specific manner. However, the effects of highly stable alkyl phosphotriester (PTE) lesions that are induced by chemical mutagens and refractory to DNA repair have not been previously studied for their effects on helicases. In this study, DNA repair and replication helicases were examined for unwinding a forked duplex DNA substrate harboring a single isopropyl PTE specifically positioned in the helicase-translocating or -nontranslocating strand within the double-stranded region. A comparison of SF2 helicases (RecQ, RECQ1, WRN, BLM, FANCJ, and ChlR1) with a SF1 DNA repair helicase (UvrD) and two replicative helicases (MCM and DnaB) demonstrates unique differences in the effect of the PTE on the DNA unwinding reactions catalyzed by these enzymes. All of the SF2 helicases tested were inhibited by the PTE lesion, whereas UvrD and the replication fork helicases were fully tolerant of the isopropyl backbone modification, irrespective of strand. Sequestration studies demonstrated that RECQ1 helicase was trapped by the PTE lesion only when it resided in the helicase-translocating strand. Our results are discussed in light of the current models for DNA unwinding by helicases that are likely to encounter sugar phosphate backbone damage during biological DNA transactions.     

MCM10 mediates RECQ4 association with MCM2‐7 helicase complex during DNA replication

Mutations in RECQ4, a member of the RecQ family of DNA helicases, have been linked to the progeroid disease Rothmund–Thomson Syndrome. Attempts to understand the complex phenotypes observed in recq4‐deficient cells suggest a potential involvement in DNA repair and replication, yet the molecular basis of the function of RECQ4 in these processes remains unknown. Here, we report the identification of a highly purified chromatin‐bound RECQ4 complex from human cell extracts. We found that essential replisome factors MCM10, MCM2‐7 helicase, CDC45 and GINS are the primary interaction partner proteins of human RECQ4. Importantly, complex formation and the association of RECQ4 with the replication origin are cell‐cycle regulated. Furthermore, we show that MCM10 is essential for the integrity of the RECQ4–MCM replicative helicase complex. MCM10 interacts directly with RECQ4 and regulates its DNA unwinding activity, and that this interaction may be modulated by cyclin‐dependent kinase phosphorylation. Thus, these studies show that RECQ4 is an integral component of the MCM replicative helicase complex participating in DNA replication in human cells.     

Stimulation of MCM helicase activity by a Cdc6 protein in the archaeon Thermoplasma acidophilum

Replicative DNA helicases are ring-shaped hexamers that play an essential role in chromosomal DNA replication. They unwind the two strands of the duplex DNA and provide the single-stranded (ss) DNA substrate for the polymerase. The minichromosome maintenance (MCM) proteins are thought to function as the replicative helicases in eukarya and archaea. The proteins of only a few archaeal organisms have been studied and revealed that although all have similar amino acid sequences and overall structures they differ in their biochemical properties. In this report the biochemical properties of the MCM protein from the archaeon Thermoplasma acidophilum is described. The enzyme has weak helicase activity on a substrate containing only a 3′-ssDNA overhang region and the protein requires a forked DNA structure for efficient helicase activity. It was also found that the helicase activity is stimulated by one of the two T.acidophilum Cdc6 homologues. This is an interesting observation as it is in sharp contrast to observations made with MCM and Cdc6 homologues from other archaea in which the helicase activity is inhibited when bound to Cdc6.     

Localization of an accessory helicase at the replisome is critical in sustaining efficient genome duplication

Genome duplication requires accessory helicases to displace proteins ahead of advancing replication forks. Escherichia coli contains three helicases, Rep, UvrD and DinG, that might promote replication of protein-bound DNA. One of these helicases, Rep, also interacts with the replicative helicase DnaB. We demonstrate that Rep is the only putative accessory helicase whose absence results in an increased chromosome duplication time. We show also that the interaction between Rep and DnaB is required for Rep to maintain rapid genome duplication. Furthermore, this Rep-DnaB interaction is critical in minimizing the need for both recombinational processing of blocked replication forks and replisome reassembly, indicating that colocalization of Rep and DnaB minimizes stalling and subsequent inactivation of replication forks. These data indicate that E. coli contains only one helicase that acts as an accessory motor at the fork in wild-type cells, that such an activity is critical for the maintenance of rapid genome duplication and that colocalization with the replisome is crucial for this function. Given that the only other characterized accessory motor, Saccharomyces cerevisiae Rrm3p, associates physically with the replisome, our demonstration of the functional importance of such an association indicates that colocalization may be a conserved feature of accessory replicative motors.     

Isolation and characterization of temperature-sensitive mutants of the Staphylococcus aureus dnaC gene

A protein encoded by the Staphylococcus aureus dnaC gene has 44% and 58% homology with Escherichia coli DnaB and Bacillus subtilis DnaC replicative DNA helicases, respectively. We identified five mutant strains whose temperature-sensitive colony formation phenotypes were complemented by the dnaC gene. DNA replication in these mutants has a fast-stop phenotype, indicating that the S. aureus dnaC gene encodes the replicative DNA helicase required for the elongation step. These mutants were also sensitive to UV irradiation, suggesting that the dnaC gene is involved in DNA repair. The number of viable mutant cells decreased at a non-permissive temperature, suggesting that S. aureus DnaC helicase is a promising target for antibiotics providing bactericidal effects.     

Mcm10 plays a role in functioning of the eukaryotic replicative DNA helicase, Cdc45-Mcm-GINS

Eukaryotic DNA replication is initiated at multiple origins of replication, where many replication proteins assemble under the control of the cell cycle [1]. A key process of replication initiation is to convert inactive Mcm2-7 to active Cdc45-Mcm-GINS (CMG) replicative helicase [2]. However, it is not known whether the CMG assembly would automatically activate its helicase activity and thus assemble the replisome. Mcm10 is an evolutionally conserved essential protein required for the initiation of replication [3, 4]. Although the roles of many proteins involved in the initiation are understood, the role of Mcm10 remains controversial [5-9]. To characterize Mcm10 in more detail, we constructed budding yeast cells bearing a degron-fused Mcm10 protein that can be efficiently degraded in response to auxin. In the absence of Mcm10, a stable CMG complex was assembled at origins. However, subsequent translocation of CMG, replication protein A loading to origins, and the intra-S checkpoint activation were severely diminished, suggesting that origin unwinding is defective. We also found that Mcm10 associates with origins during initiation in an S-cyclin-dependent kinase- and Cdc45-dependent manner. Thus, Mcm10 plays an essential role in functioning of the CMG replicative helicase independent of assembly of a stable CMG complex at origins.     

Atm deficiency affects both apoptosis and proliferation to augment Myc-induced lymphomagenesis

Myc oncoproteins are commonly activated in malignancies and are sufficient to provoke many types of cancer. However, the critical mechanisms by which Myc contributes to malignant transformation are not clear. DNA damage seems to be an important initiating event in tumorigenesis. Here, we show that although Myc does not directly induce double-stranded DNA breaks, it does augment activation of the Atm/p53 DNA damage response pathway, suggesting that Atm may function as a guardian against Myc-induced transformation. Indeed, we show that Atm loss augments Myc-induced lymphomagenesis and impairs Myc-induced apoptosis, which normally harnesses Myc-driven tumorigenesis. Surprisingly, Atm loss also augments the proliferative response induced by Myc, and this augmentation is associated with enhanced suppression of the expression of the cyclin-dependent kinase inhibitor p27(Kip1). Therefore, regulation of cell proliferation and p27(Kip1) seems to be a contributing mechanism by which Atm holds tumor formation in check.     

Replicative helicase loaders: ring breakers and ring makers

Helicase loaders transfer the ring-shaped replicative helicases onto DNA. They assort into two classes: ring breakers, which place stabile hexameric rings on DNA via transient gaps at subunit interfaces; and helicase makers, which assemble hexameric rings around DNA from monomeric helicase units.     

The replicative helicases of bacteria, archaea, and eukarya can unwind RNA-DNA hybrid substrates

Replicative helicases are hexameric enzymes that unwind DNA during chromosomal replication. They use energy from nucleoside triphosphate hydrolysis to translocate along one strand of the duplex DNA and displace the complementary strand. Here, the ability of a replicative helicase from each of the three domains, bacteria, archaea, and eukarya, to unwind RNA-containing substrate was determined. It is shown that all three helicases can unwind DNA-RNA hybrids while translocating along the single-stranded DNA. No unwinding could be observed when the helicases were provided with a single-stranded RNA overhang. Using DNA, RNA, and DNA-RNA chimeric oligonucleotides it was found that whereas the enzymes can bind both DNA and RNA, they could translocate only along DNA and only DNA stimulates the ATPase activity of the enzymes. Recent observations suggest that helicases may interact with enzymes participating in RNA metabolism and that RNA-DNA hybrids may be present on the chromosomes. Thus, the results presented here may suggest a new role for the replicative helicases during chromosomal replication or in other cellular processes.     

High-temperature single-molecule kinetic analysis of thermophilic archaeal MCM helicases

The minichromosome maintenance (MCM) complex is the replicative helicase responsible for unwinding DNA during archaeal and eukaryal genome replication. To mimic long helicase events in the cell, a high-temperature single-molecule assay was designed to quantitatively measure long-range DNA unwinding of individual DNA helicases from the archaeons Methanothermobacter thermautotrophicus (Mth) and Thermococcus sp. 9°N (9°N). Mth encodes a single MCM homolog while 9°N encodes three helicases. 9°N MCM3, the proposed replicative helicase, unwinds DNA at a faster rate compared to 9°N MCM2 and to Mth MCM. However, all three MCM proteins have similar processivities. The implications of these observations for DNA replication in archaea and the differences and similarities among helicases from different microorganisms are discussed. Development of the high-temperature single-molecule assay establishes a system to comprehensively study thermophilic replisomes and evolutionary links between archaeal, eukaryal, and bacterial replication systems.     

RecQ4: the second replicative helicase?

Recent work has greatly contributed to the understanding of the biology and biochemistry of RecQ4. It plays an essential non-enzymatic role in the formation of the CMG complex, and thus replication initiation, by means of its Sld2 homologous domain. The helicase domain of RecQ4 has now been demonstrated to possess 3′–5′ DNA helicase activity, like the other members of the RecQ family. The biological purpose of this activity is still unclear, but helicase-dead mutants are unable to restore viability in the absence of wildtype RecQ4. This indicates that RecQ4 performs a second role, which requires helicase activity and is implicated in replication and DNA repair. Thus, it is clear that two helicases, RecQ4 and Mcm2-7, are integral to replication. The nature of the simultaneous involvement of these two helicases remains to be determined, and possible models will be proposed.