The Replication Initiation Protein Sld2 Regulates Helicase Assembly

Assembly of the Cdc45-Mcm2-7-GINS (CMG) replicative helicase complex must be regulated to ensure that DNA unwinding is coupled with DNA synthesis. Sld2 is required for the initiation of DNA replication in budding yeast. We identified a mutant of Sld2, Sld2-m1,4, that is specifically defective in Mcm2-7 binding. When this sld2-m1,4 mutant is expressed, cells exhibit severe inhibition of DNA replication. Furthermore, the CMG complex assembles prematurely in G₁ in mutant cells, but not wild-type cells. These data suggest that Sld2 binding to Mcm2-7 is essential to block the inappropriate formation of a CMG helicase complex in G₁. We also study a mutant of Sld2 that is defective in binding DNA, sld2-DNA, and find that sld2-DNA cells exhibit no GINS-Mcm2-7 interaction. These data suggest that Sld2 association with DNA is required for CMG assembly in S phase.     

TbPIF1, a Trypanosoma brucei Mitochondrial DNA Helicase, Is Essential for Kinetoplast Minicircle Replication

Kinetoplast DNA, the trypanosome mitochondrial genome, is a network of interlocked DNA rings including several thousand minicircles and a few dozen maxicircles. Minicircles replicate after release from the network, and their progeny reattach. Remarkably, trypanosomes have six mitochondrial DNA helicases related to yeast PIF1 helicase. Here we report that one of the six, TbPIF1, functions in minicircle replication. RNA interference (RNAi) of TbPIF1 causes a growth defect and kinetoplast DNA loss. Minicircle replication intermediates decrease during RNAi, and there is an accumulation of multiply interlocked, covalently closed minicircle dimers (fraction U). In studying the significance of fraction U, we found that this species also accumulates during RNAi of mitochondrial topoisomerase II. These data indicate that one function of TbPIF1 is an involvement, together with topoisomerase II, in the segregation of minicircle progeny.     

An Interaction between DNA Polymerase and Helicase Is Essential for the High Processivity of the Bacteriophage T7 Replisome

Synthesis of the leading DNA strand requires the coordinated activity of DNA polymerase and DNA helicase, whereas synthesis of the lagging strand involves interactions of these proteins with DNA primase. We present the first structural model of a bacteriophage T7 DNA helicase-DNA polymerase complex using a combination of small angle x-ray scattering, single-molecule, and biochemical methods. We propose that the protein-protein interface stabilizing the leading strand synthesis involves two distinct interactions: a stable binding of the helicase to the palm domain of the polymerase and an electrostatic binding of the carboxyl-terminal tail of the helicase to a basic patch on the polymerase. DNA primase facilitates binding of DNA helicase to ssDNA and contributes to formation of the DNA helicase-DNA polymerase complex by stabilizing DNA helicase.     

GINS and Sld3 compete with one another for Mcm2-7 and Cdc45 binding

Sld3 is essential for the initiation of DNA replication, but Sld3 does not travel with a replication fork. GINS binds to Cdc45 and Mcm2-7 to form the replication fork helicase in eukaryotes. We purified Sld3, Cdc45, GINS, and Mcm2-7 and studied their interaction and assembly into complexes. Sld3 binds tightly to Cdc45 in the presence or absence of cyclin-dependent kinase activity. Furthermore, Sld3 binds tightly to the Mcm2-7 complex, and a ternary complex forms among Cdc45, Mcm2-7, and Sld3, with a 1:1:1 stoichiometry (CMS complex). GINS binds directly to Mcm2-7, and GINS competes with Sld3 for Mcm2-7 binding. GINS also binds directly to Cdc45, and GINS competes with Sld3 for Cdc45 binding. Cdc45, Mcm2-7, and GINS form a ternary complex with a stoichiometry of 1:1:1 (CMG complex). Size exclusion data reveal that when Sld3, Cdc45, Mcm2-7, and GINS are added together, the result is a mixture of CMS and CMG complexes. The data suggest that GINS and Sld3 compete with one another for Mcm2-7 and Cdc45 binding. Our results are consistent with a model wherein GINS trades places with Sld3 at a replication origin, contributing to the activation of the replication fork helicase.     

Enhancement of Human DNA Polymerase η Activity and Fidelity Is Dependent Upon a Bipartite Interaction with the Werner Syndrome Protein

We have investigated the interaction between human DNA polymerase η (hpol η) and the Werner syndrome protein (WRN). Functional assays revealed that the WRN exonuclease and RecQ C-terminal (RQC) domains are necessary for full stimulation of hpol η-catalyzed formation of correct base pairs. We find that WRN does not stimulate hpol η-catalyzed formation of mispairs. Moreover, the exonuclease activity of WRN prevents stable mispair formation by hpol η. These results are consistent with a proofreading activity for WRN during single-nucleotide additions. ATP hydrolysis by WRN appears to attenuate stimulation of hpol η. Pre-steady-state kinetic results show that k_pol is increased 4-fold by WRN. Finally, pulldown assays reveal a bipartite physical interaction between hpol η and WRN that is mediated by the exonuclease and RQC domains. Taken together, these results are consistent with alteration of the rate-limiting step in polymerase catalysis by direct protein-protein interactions between WRN and hpol η. In summary, WRN improves the efficiency and fidelity of hpol η to promote more effective replication of DNA.     

Control of Helicase Loading in the Coupled DNA Replication and Recombination Systems of Bacteriophage T4

The Gp59 protein of bacteriophage T4 promotes DNA replication by loading the replicative helicase, Gp41, onto replication forks and recombination intermediates. Gp59 also blocks DNA synthesis by Gp43 polymerase until Gp41 is loaded, ensuring that synthesis is tightly coupled to unwinding. The distinct polymerase blocking and helicase loading activities of Gp59 likely involve different binding interactions with DNA and protein partners. Here, we investigate how interactions of Gp59 with DNA and Gp32, the T4 single-stranded DNA (ssDNA)-binding protein, are related to these activities. A previously characterized mutant, Gp59-I87A, exhibits markedly reduced affinity for ssDNA and pseudo-fork DNA substrates. We demonstrate that on Gp32-covered ssDNA, the DNA binding defect of Gp59-I87A is not detrimental to helicase loading and translocation. In contrast, on pseudo-fork DNA the I87A mutation is detrimental to helicase loading and unwinding in the presence or absence of Gp32. Other results indicate that Gp32 binding to lagging strand ssDNA relieves the blockage of Gp43 polymerase activity by Gp59, whereas the inhibition of Gp43 exonuclease activity is maintained. Our findings suggest that Gp59-Gp32 and Gp59-DNA interactions perform separate but complementary roles in T4 DNA metabolism; Gp59-Gp32 interactions are needed to load Gp41 onto D-loops, and other nucleoprotein structures containing clusters of Gp32. Gp59-DNA interactions are needed to load Gp41 onto nascent or collapsed replication forks lacking clusters of Gp32 and to coordinate bidirectional replication from T4 origins. The dual functionalities of Gp59 allow it to promote the initiation or re-start of DNA replication from a wide variety of recombination and replication intermediates.     

Protein Phosphatase 2A and Cdc7 Kinase Regulate the DNA Unwinding Element-binding Protein in Replication Initiation

The DNA unwinding element (DUE)-binding protein (DUE-B) binds to replication origins coordinately with the minichromosome maintenance (MCM) helicase and the helicase activator Cdc45 in vivo, and loads Cdc45 onto chromatin in Xenopus egg extracts. Human DUE-B also retains the aminoacyl-tRNA proofreading function of its shorter orthologs in lower organisms. Here we report that phosphorylation of the DUE-B unstructured C-terminal domain unique to higher organisms regulates DUE-B intermolecular binding. Gel filtration analyses show that unphosphorylated DUE-B forms multiple high molecular weight (HMW) complexes. Several aminoacyl-tRNA synthetases and Mcm2–7 proteins were identified by mass spectrometry of the HMW complexes. Aminoacyl-tRNA synthetase binding is RNase A sensitive, whereas interaction with Mcm2–7 is nuclease resistant. Unphosphorylated DUE-B HMW complex formation is decreased by PP2A inhibition or direct DUE-B phosphorylation, and increased by inhibition of Cdc7. These results indicate that the state of DUE-B phosphorylation is maintained by the equilibrium between Cdc7-dependent phosphorylation and PP2A-dependent dephosphorylation, each previously shown to regulate replication initiation. Alanine mutation of the DUE-B C-terminal phosphorylation target sites increases MCM binding but blocks Cdc45 loading in vivo and inhibits cell division. In egg extracts alanine mutation of the DUE-B C-terminal phosphorylation sites blocks Cdc45 loading and inhibits DNA replication. The effects of DUE-B C-terminal phosphorylation reveal a novel S phase kinase regulatory mechanism for Cdc45 loading and MCM helicase activation.     

The N-terminal Domain of the Drosophila Mitochondrial Replicative DNA Helicase Contains an Iron-Sulfur Cluster and Binds DNA

The metazoan mitochondrial DNA helicase is an integral part of the minimal mitochondrial replisome. It exhibits strong sequence homology with the bacteriophage T7 gene 4 protein primase-helicase (T7 gp4). Both proteins contain distinct N- and C-terminal domains separated by a flexible linker. The C-terminal domain catalyzes its characteristic DNA-dependent NTPase activity, and can unwind duplex DNA substrates independently of the N-terminal domain. Whereas the N-terminal domain in T7 gp4 contains a DNA primase activity, this function is lost in metazoan mtDNA helicase. Thus, although the functions of the C-terminal domain and the linker are partially understood, the role of the N-terminal region in the metazoan replicative mtDNA helicase remains elusive. Here, we show that the N-terminal domain of Drosophila melanogaster mtDNA helicase coordinates iron in a 2Fe-2S cluster that enhances protein stability in vitro. The N-terminal domain binds the cluster through conserved cysteine residues (Cys⁶⁸, Cys⁷¹, Cys¹⁰², and Cys¹⁰⁵) that are responsible for coordinating zinc in T7 gp4. Moreover, we show that the N-terminal domain binds both single- and double-stranded DNA oligomers, with an apparent K_d of ∼120 nm. These findings suggest a possible role for the N-terminal domain of metazoan mtDNA helicase in recruiting and binding DNA at the replication fork.     

The Full-length Saccharomyces cerevisiae Sgs1 Protein Is a Vigorous DNA Helicase That Preferentially Unwinds Holliday Junctions

The highly conserved RecQ family of DNA helicases has multiple roles in the maintenance of genome stability. Sgs1, the single RecQ homologue in Saccharomyces cerevisiae, acts both early and late during homologous recombination. Here we present the expression, purification, and biochemical analysis of full-length Sgs1. Unlike the truncated form of Sgs1 characterized previously, full-length Sgs1 binds diverse single-stranded and double-stranded DNA substrates, including DNA duplexes with 5′- and 3′-single-stranded DNA overhangs. Similarly, Sgs1 unwinds a variety of DNA substrates, including blunt-ended duplex DNA. Significantly, a substrate containing a Holliday junction is unwound most efficiently. DNA unwinding is catalytic, requires ATP, and is stimulated by replication protein A. Unlike RecQ homologues from multicellular organisms, Sgs1 is remarkably active at picomolar concentrations and can efficiently unwind duplex DNA molecules as long as 23,000 base pairs. Our analysis shows that Sgs1 resembles Escherichia coli RecQ protein more than any of the human RecQ homologues with regard to its helicase activity. The full-length recombinant protein will be invaluable for further investigation of Sgs1 biochemistry.     

Helicase Subunit Cdc45 Targets the Checkpoint Kinase Rad53 to Both Replication Initiation and Elongation Complexes after Fork Stalling

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The C-terminal 50 Amino Acid Residues of Dengue NS3 Protein Are Important for NS3-NS5 Interaction and Viral Replication

Dengue virus multifunctional proteins NS3 protease/helicase and NS5 methyltransferase/RNA-dependent RNA polymerase form part of the viral replication complex and are involved in viral RNA genome synthesis, methylation of the 5′-cap of viral genome, and polyprotein processing among other activities. Previous studies have shown that NS5 residue Lys-330 is required for interaction between NS3 and NS5. Here, we show by competitive NS3-NS5 interaction ELISA that the NS3 peptide spanning residues 566–585 disrupts NS3-NS5 interaction but not the null-peptide bearing the N570A mutation. Small angle x-ray scattering study on NS3(172–618) helicase and covalently linked NS3(172–618)-NS5(320–341) reveals a rigid and compact formation of the latter, indicating that peptide NS5(320–341) engages in specific and discrete interaction with NS3. Significantly, NS3:Asn-570 to alanine mutation introduced into an infectious DENV2 cDNA clone did not yield detectable virus by plaque assay even though intracellular double-stranded RNA was detected by immunofluorescence. Detection of increased negative-strand RNA synthesis by real time RT-PCR for the NS3:N570A mutant suggests that NS3-NS5 interaction plays an important role in the balanced synthesis of positive- and negative-strand RNA for robust viral replication. Dengue virus infection has become a global concern, and the lack of safe vaccines or antiviral treatments urgently needs to be addressed. NS3 and NS5 are highly conserved among the four serotypes, and the protein sequence around the pinpointed amino acids from the NS3 and NS5 regions are also conserved. The identification of the functionally essential interaction between the two proteins by biochemical and reverse genetics methods paves the way for rational drug design efforts to inhibit viral RNA synthesis.     

The DDN Catalytic Motif Is Required for Metnase Functions in Non-homologous End Joining (NHEJ) Repair and Replication Restart

Metnase (or SETMAR) arose from a chimeric fusion of the Hsmar1 transposase downstream of a protein methylase in anthropoid primates. Although the Metnase transposase domain has been largely conserved, its catalytic motif (DDN) differs from the DDD motif of related transposases, which may be important for its role as a DNA repair factor and its enzymatic activities. Here, we show that substitution of DDN⁶¹⁰ with either DDD⁶¹⁰ or DDE⁶¹⁰ significantly reduced in vivo functions of Metnase in NHEJ repair and accelerated restart of replication forks. We next tested whether the DDD or DDE mutants cleave single-strand extensions and flaps in partial duplex DNA and pseudo-Tyr structures that mimic stalled replication forks. Neither substrate is cleaved by the DDD or DDE mutant, under the conditions where wild-type Metnase effectively cleaves ssDNA overhangs. We then characterized the ssDNA-binding activity of the Metnase transposase domain and found that the catalytic domain binds ssDNA but not dsDNA, whereas dsDNA binding activity resides in the helix-turn-helix DNA binding domain. Substitution of Asn-610 with either Asp or Glu within the transposase domain significantly reduces ssDNA binding activity. Collectively, our results suggest that a single mutation DDN⁶¹⁰ → DDD⁶¹⁰, which restores the ancestral catalytic site, results in loss of function in Metnase.     

Cdc45 Is a Critical Effector of Myc-Dependent DNA Replication Stress

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Disease Variants of the Human Mitochondrial DNA Helicase Encoded by C10orf2 Differentially Alter Protein Stability,Nucleotide Hydrolysis,and Helicase Activity

Missense mutations in the human C10orf2 gene, encoding the mitochondrial DNA (mtDNA) helicase, co-segregate with mitochondrial diseases such as adult-onset progressive external ophthalmoplegia, hepatocerebral syndrome with mtDNA depletion syndrome, and infantile-onset spinocerebellar ataxia. To understand the biochemical consequences of C10orf2 mutations, we overproduced wild type and 20 mutant forms of human mtDNA helicase in Escherichia coli and developed novel schemes to purify the recombinant enzymes to near homogeneity. A combination of molecular crowding, non-ionic detergents, Mg²⁺ ions, and elevated ionic strength was required to combat insolubility and intrinsic instability of certain mutant variants. A systematic biochemical assessment of the enzymes included analysis of DNA binding affinity, DNA helicase activity, the kinetics of nucleotide hydrolysis, and estimates of thermal stability. In contrast to other studies, we found that all 20 mutant variants retain helicase function under optimized in vitro conditions despite partial reductions in DNA binding affinity, nucleotide hydrolysis, or thermal stability for some mutants. Such partial defects are consistent with the delayed presentation of mitochondrial diseases associated with mutation of C10orf2.     

The Dbf4-Cdc7 Kinase Promotes Mcm2-7 Ring Opening to Allow for Single-stranded DNA Extrusion and Helicase Assembly

The replication fork helicase in eukaryotes is composed of Cdc45, Mcm2-7, and GINS (CMG). The Dbf4-Cdc7 kinase phosphorylates Mcm2 in vitro, but the in vivo role for Dbf4-Cdc7 phosphorylation of Mcm2 is unclear. We find that budding yeast Dbf4-Cdc7 phosphorylates Mcm2 in vivo under normal conditions during S phase. Inhibiting Dbf4-Cdc7 phosphorylation of Mcm2 confers a dominant-negative phenotype with a severe growth defect. Inhibiting Dbf4-Cdc7 phosphorylation of Mcm2 under wild-type expression conditions also results in impaired DNA replication, substantially decreased single-stranded formation at an origin, and markedly disrupted interaction between GINS and Mcm2-7 during S phase. In vitro, Dbf4-Cdc7 kinase (DDK) phosphorylation of Mcm2 substantially weakens the interaction between Mcm2 and Mcm5, and Dbf4-Cdc7 phosphorylation of Mcm2 promotes Mcm2-7 ring opening. The extrusion of ssDNA from the central channel of Mcm2-7 triggers GINS attachment to Mcm2-7. Thus, Dbf4-Cdc7 phosphorylation of Mcm2 may open the Mcm2-7 ring at the Mcm2-Mcm5 interface, allowing for single-stranded DNA extrusion and subsequent GINS assembly with Mcm2-7.     

MCM2-7 form double hexamers at licensed origins in Xenopus egg extract

In late mitosis and G1, Mcm2-7 are assembled onto replication origins to license them for initiation in the upcoming S phase. After initiation, Mcm2-7 provide helicase activity to unwind DNA at the replication fork. Here we examine the structure of Mcm2-7 on chromatin in Xenopus egg extracts. We show that prior to replication initiation, Mcm2-7 is present at licensed replication origins in a complex with a molecular mass close to double that of the Mcm2-7 hexamer. This complex has approximately stoichiometric quantities of the 6 Mcm2-7 proteins and we conclude that it consists of a double heterohexamer. This provides a configuration potentially capable of initiating a pair of bidirectional replication forks in S phase. We also show that after initiation, Mcm2-7 associate with Cdc45 and GINS to form a relatively stable CMG (Cdc45-MCM-GINS) complex. The CMG proteins also associate less strongly with other replication proteins, consistent with the idea that a single CMG complex forms the core of the replisome.     

Rad18-mediated Translesion Synthesis of Bulky DNA Adducts Is Coupled to Activation of the Fanconi Anemia DNA Repair Pathway

Fanconi anemia (FA) is a cancer susceptibility syndrome characterized by sensitivity to DNA-damaging agents. The FA proteins (FANCs) are implicated in DNA repair, although the precise mechanisms by which FANCs process DNA lesions are not fully understood. An epistatic relationship between the FA pathway and translesion synthesis (TLS, a post-replication DNA repair mechanism) has been suggested, but the basis for cross-talk between the FA and TLS pathways is poorly understood. We show here that ectopic overexpression of the E3 ubiquitin ligase Rad18 (a central regulator of TLS) induces DNA damage-independent mono-ubiquitination of proliferating cell nuclear antigen (PCNA) (a known Rad18 substrate) and FANCD2. Conversely, DNA damage-induced mono-ubiquitination of both PCNA and FANCD2 is attenuated in Rad18-deficient cells, demonstrating that Rad18 contributes to activation of the FA pathway. WT Rad18 but not an E3 ubiquitin ligase-deficient Rad18 C28F mutant fully complements both PCNA ubiquitination and FANCD2 activation in Rad18-depleted cells. Rad18-induced mono-ubiquitination of FANCD2 is not observed in FA core complex-deficient cells, demonstrating that Rad18 E3 ligase activity alone is insufficient for FANCD2 ubiquitylation. Instead, Rad18 promotes FA core complex-dependent FANCD2 ubiquitination in a manner that is secondary to PCNA mono-ubiquitination. Taken together, these results demonstrate a novel Rad18-dependent mechanism that couples activation of the FA pathway with TLS.     

Novel Function of the Fanconi Anemia Group J or RECQ1 Helicase to Disrupt Protein-DNA Complexes in a Replication Protein A-stimulated Manner

Understanding how cellular machinery deals with chromosomal genome complexity is an important question because protein bound to DNA may affect various cellular processes of nucleic acid metabolism. DNA helicases are at the forefront of such processes, yet there is only limited knowledge how they remodel protein-DNA complexes and how these mechanisms are regulated. We have determined that representative human RecQ and Fe-S cluster DNA helicases are potently blocked by a protein-DNA interaction. The Fanconi anemia group J (FANCJ) helicase partners with the single-stranded DNA-binding protein replication protein A (RPA) to displace BamHI-E111A bound to duplex DNA in a specific manner. Protein displacement was dependent on the ATPase-driven function of the helicase and unique properties of RPA. Further biochemical studies demonstrated that the shelterin proteins TRF1 and TRF2, which preferentially bind the telomeric repeat found at chromosome ends, effectively block FANCJ from unwinding the forked duplex telomeric substrate. RPA, but not the Escherichia coli single-stranded DNA-binding protein or shelterin factor Pot1, stimulated FANCJ ejection of TRF1 from the telomeric DNA substrate. FANCJ was also able to displace TRF2 from the telomeric substrate in an RPA-dependent manner. The stimulation of helicase-catalyzed protein displacement is also observed with the DNA helicase RECQ1, suggesting a conserved functional interaction of RPA-interacting helicases. These findings suggest that partnerships between RPA and interacting human DNA helicases may greatly enhance their ability to dislodge proteins bound to duplex DNA, an activity that is likely to be highly relevant to their biological roles in DNA metabolism.     

The Involvement of Acidic Nucleoplasmic DNA-binding Protein (And-1) in the Regulation of Prereplicative Complex (pre-RC) Assembly in Human Cells

DNA replication in all eukaryotes starts with the process of loading the replicative helicase MCM2–7 onto chromatin during late mitosis of the cell cycle. MCM2–7 is a key component of the prereplicative complex (pre-RC), which is loaded onto chromatin by the concerted action of origin recognition complex, Cdc6, and Cdt1. Here, we demonstrate that And-1 is assembled onto chromatin in late mitosis and early G₁ phase before the assembly of pre-RC in human cells. And-1 forms complexes with MCM2–7 to facilitate the assembly of MCM2–7 onto chromatin at replication origins in late mitosis and G₁ phase. We also present data to show that depletion of And-1 significantly reduces the interaction between Cdt1 and MCM7 in G₁ phase cells. Thus, human And-1 facilitates loading of the MCM2–7 helicase onto chromatin during the assembly of pre-RC.