Evolution of Lambdoid Replication Modules

Comparison of the putative iteron-binding proteins of lambdoid phages allows us to propose that in the case of lambdoid replication modules, the units on which natural selection acts do not coincide with the open reading frames. Rather, the first replication gene is split into two segments, and its 3 part (corresponding to the C-terminal domain of the iteron-binding protein) forms one unit with the second gene. We also propose from the phylogenetic analysis of phage-encoded homologs of E. coli DnaB and DnaC, that the recombination with the host sequences is not frequent. Accessory ATP-ases for helicase loading (E. coli DnaC homologs) may not be universal replication proteins. Our analysis may suggest that the bacterial helicase loaders might be of phage origin. The comparison of DnaC homologs of enterobacteria and enterobacterial phages supports the experimental data on residues important in interaction with DnaB. We propose that construction of plasmids carrying the replication origins of lambdoid prophages could be useful not only in further research on DNA replication but also on the role of these prophages in shuttling genes for bacterial virulence. The phage replication sequences could be also useful for identification of clinical enterobacterial isolates.     

Rolling circle replication of human papillomavirus type 16 DNA in epithelial cell extracts

Replication of human papillomavirus (HPV) genomes requires an origin of replication and two viral proteins: the DNA helicase E1 and the auxiliary factor E2. To dissect the profile of HPV replication in the epithelium, we analyzed replication of an HPV16 origin-containing plasmid in human epithelial cell extracts supplemented with purified E1 and E2. We found that in addition to well-defined circular replication products, high-molecular-weight DNA was synthesized in a manner that depended on the origin, E1 and E2. The high-molecular-weight DNA was converted to a unit-length linear DNA by treatment with restriction enzymes that cleave the plasmid once, implying that a concatemeric DNA was generated by rolling circle replication. Nicking or relaxing the template plasmid enhanced the level of HPV rolling circle replication. In contrast, the addition of an extract from non-epithelial cells diminished the generation of the rolling circle replication product in the epithelial cell extract, indicating factors that counteract HPV rolling circle replication. These results suggest a rolling circle replication mechanism for the HPV genome in cervical epithelial cells, which may have physiological implications for generation of the tandem-repeated HPV genomes occasionally found integrated into the chromosome of cervical cancer cells.     

Nbs1-dependent binding of Mre11 to adenovirus E4 mutant viral DNA is important for inhibiting DNA replication

Adenovirus (Ad) infections stimulate the activation of cellular DNA damage response and repair pathways. Ad early regulatory proteins prevent activation of DNA damage responses by targeting the MRN complex, composed of the Mre11, Rad50 and Nbs1 proteins, for relocalization and degradation. In the absence of these viral proteins, Mre11 colocalizes with viral DNA replication foci. Mre11 foci formation at DNA damage induced by ionizing radiation depends on the Nbs1 component of the MRN complex and is stabilized by the mediator of DNA damage checkpoint protein 1 (Mdc1). We find that Nbs1 is required for Mre11 localization at DNA replication foci in Ad E4 mutant infections. Mre11 is important for Mdc1 foci formation in infected cells, consistent with its role as a sensor of DNA damage. Chromatin immunoprecipitation assays indicate that both Mre11 and Mdc1 are physically bound to viral DNA, which could account for their localization in viral DNA containing foci. Efficient binding of Mre11 to E4 mutant DNA depends on the presence of Nbs1, and is correlated with a significant E4 mutant DNA replication defect. Our results are consistent with a model in which physical interaction of Mre11 with viral DNA is mediated by Nbs1, and interferes with viral DNA replication.     

The X-ray structure of the papillomavirus helicase in complex with its molecular matchmaker E2

DNA replication of the papillomaviruses is specified by cooperative binding of two proteins to the ori site: the enhancer E2 and the viral initiator E1, a distant member of the AAA+ family of proteins. Formation of this prereplication complex is an essential step toward the construction of a functional, multimeric E1 helicase and DNA melting. To understand how E2 interacts with E1 to regulate this process, we have solved the X-ray structure of a complex containing the HPV18 E2 activation domain bound to the helicase domain of E1. Modeling the monomers of E1 to a hexameric helicase shows that E2 blocks hexamerization of E1 by shielding a region of the E1 oligomerization surface and stabilizing a conformation of E1 that is incompatible with ATP binding. Further biochemical experiments and structural analysis show that ATP is an allosteric effector of the dissociation of E2 from E1. Our data provide the first molecular insights into how a protein can regulate the assembly of an oligomeric AAA+ complex and explain at a structural level why E2, after playing a matchmaker role by guiding E1 to the DNA, must dissociate for subsequent steps of initiation to occur. Building on previously proposed ideas, we discuss how our data advance current models for the conversion of E1 in the prereplication complex to a hexameric helicase assembly.     

A carboxyl-terminal serine of the bovine papillomavirus E1 protein is phosphorylated in vivo and in vitro

The E1 protein of bovine papillomavirus (BPV) plays several key roles in viral DNA replication. E1 binds the viral origin, unwinds template DNA at the replication fork and recruits cellular replication machinery to the viral DNA. E1 is phosphorylated at multiple sites, and phosphorylation of E1 regulates E1 function and viral DNA replication. A consensus motif for the cellular kinase CK2 was identified at serine 584 near the carboxyl terminus of BPV E1, and found to be highly conserved among papillomavirus E1 proteins. Serine 584 was identified as a substrate of CK1 and CK2 in vitro by mutational and biochemical analysis, and was phosphorylated by a cellular kinase in cultured cells.     

Structural and mechanistic insights into Mcm2–7 double-hexamer assembly and function

Eukaryotic cells license each DNA replication origin during G1 phase by assembling a prereplication complex that contains a Mcm2–7 (minichromosome maintenance proteins 2–7) double hexamer. During S phase, each Mcm2–7 hexamer forms the core of a replicative DNA helicase. However, the mechanisms of origin licensing and helicase activation are poorly understood. The helicase loaders ORC–Cdc6 function to recruit a single Cdt1–Mcm2–7 heptamer to replication origins prior to Cdt1 release and ORC–Cdc6–Mcm2–7 complex formation, but how the second Mcm2–7 hexamer is recruited to promote double-hexamer formation is not well understood. Here, structural evidence for intermediates consisting of an ORC–Cdc6–Mcm2–7 complex and an ORC–Cdc6–Mcm2–7–Mcm2–7 complex are reported, which together provide new insights into DNA licensing. Detailed structural analysis of the loaded Mcm2–7 double-hexamer complex demonstrates that the two hexamers are interlocked and misaligned along the DNA axis and lack ATP hydrolysis activity that is essential for DNA helicase activity. Moreover, we show that the head-to-head juxtaposition of the Mcm2–7 double hexamer generates a new protein interaction surface that creates a multisubunit-binding site for an S-phase protein kinase that is known to activate DNA replication. The data suggest how the double hexamer is assembled and how helicase activity is regulated during DNA licensing, with implications for cell cycle control of DNA replication and genome stability.     

Development of quantitative and high-throughput assays of polyomavirus and papillomavirus DNA replication

Polyoma- and papillomaviruses genome replication is initiated by the binding of large T antigen (LT) and of E1 and E2, respectively, at the viral origin (ori). Replication of an ori-containing plasmid occurs in cells transiently expressing these viral proteins and is typically quantified by Southern blotting or PCR. To facilitate the study of SV40 and HPV31 DNA replication, we developed cellular assays in which transient replication of the ori-plasmid is quantified using a firefly luciferase gene located in cis to the ori. Under optimized conditions, replication of the SV40 and HPV31 ori-plasmids resulted in a 50- and 150-fold increase in firefly luciferase levels, respectively. These results were validated using replication-defective mutants of LT, E1 and E2 and with inhibitors of DNA replication and cell-cycle progression. These quantitative and high-throughput assays should greatly facilitate the study of SV40 and HPV31 DNA replication and the identification of small-molecule inhibitors of this process.     

Concerted activities of Mcm4, Sld3, and Dbf4 in control of origin activation and DNA replication fork progression

Eukaryotic chromosomes initiate DNA synthesis from multiple replication origins in a temporally specific manner during S phase. The replicative helicase Mcm2-7 functions in both initiation and fork progression and thus is an important target of regulation. Mcm4, a helicase subunit, possesses an unstructured regulatory domain that mediates control from multiple kinase signaling pathways, including the Dbf4-dependent Cdc7 kinase (DDK). Following replication stress in S phase, Dbf4 and Sld3, an initiation factor and essential target of Cyclin-Dependent Kinase (CDK), are targets of the checkpoint kinase Rad53 for inhibition of initiation from origins that have yet to be activated, so-called late origins. Here, whole-genome DNA replication profile analysis is used to access under various conditions the effect of mutations that alter the Mcm4 regulatory domain and the Rad53 targets, Sld3 and Dbf4. Late origin firing occurs under genotoxic stress when the controls on Mcm4, Sld3, and Dbf4 are simultaneously eliminated. The regulatory domain of Mcm4 plays an important role in the timing of late origin firing, both in an unperturbed S phase and in dNTP limitation. Furthermore, checkpoint control of Sld3 impacts fork progression under replication stress. This effect is parallel to the role of the Mcm4 regulatory domain in monitoring fork progression. Hypomorph mutations in sld3 are suppressed by a mcm4 regulatory domain mutation. Thus, in response to cellular conditions, the functions executed by Sld3, Dbf4, and the regulatory domain of Mcm4 intersect to control origin firing and replication fork progression, thereby ensuring genome stability.Eukaryotic cells initiate DNA synthesis from multiple replication origins on each chromosome to ensure efficient duplication of the genome in S phase. Activation of replication origins is achieved through two distinct steps that take place at separate stages of the cell division cycle. The first step, licensing of replication origins, occurs in G1 when CDK activity is low (Diffley 2011). During this process, a double hexameric minichromosome maintenance (MCM) complex, composed of two Mcm2-7 hexamers, is loaded onto each replication origin to form a pre-Replicative Complex (pre-RC) by the Origin Recognition Complex (ORC) and licensing factors, Cdc6 and Cdt1 (encoded by the TAH11 gene) (Diffley 2011). The second step, activation of licensed origins, occurs at each origin in a temporally controlled manner throughout S phase and requires activities of two S phase kinases, the S phase Cyclin-dependent Kinases (CDKs), and the Dbf4-dependent Cdc7 kinase (DDK) (Tanaka and Araki 2013). CDK phosphorylates two key substrates, Sld2 and Sld3, and promotes their binding to Dpb11 (Tanaka et al. 2007; Zegerman and Diffley 2007). DDK phosphorylates several subunits of the Mcm2-7 hexamer and, most importantly, blocks an intrinsic inhibitory activity residing within the amino-terminus of the Mcm4 subunit (Sheu and Stillman 2006, 2010; Randell et al. 2010). The action of these S phase kinases facilitates recruitment of Cdc45 and the GINS complex, composed of protein subunits Sld5, Psf1, Psf2, and Psf3, to the inactive MCM double hexamer and converts it into an active helicase complex, composed of Cdc45, Mcm2-7, and GINS (the CMG complex) (Tanaka and Araki 2013). The two-step process separates the loading and activation of replicative helicases at origins and thereby ensures that initiation from each origin occurs once and only once during each cell division cycle. Once origins are fully activated, the double helix unwinds, and DNA polymerase and other replisome components are recruited to establish replication forks, where new DNA is copied bidirectionally from each origin.Initiation of DNA synthesis from licensed origins across the genome (origin firing) follows a predetermined temporal pattern (Rhind and Gilbert 2013). In budding yeast, the timing of DNA replication can be traced to the activation of individual origins. Origin activation occurs continuously during S phase, but those that fire first in S phase are referred to as early origins, and those that fire later are late origins. Despite being an essential target of CDK, Sld3, together with Sld7 and Cdc45, binds to the loaded Mcm2-7 hexamer in a manner dependent on DDK but not CDK (Heller et al. 2011; Tanaka et al. 2011). This association is a prerequisite for the subsequent CDK-dependent recruitment of a preloading complex, composed of Sld2, Dpb11, GINS, and pol ε (Muramatsu et al. 2010). It was proposed that DDK-dependent recruitment of the limiting Sld3-Sld7-Cdc45 is a key step for determining the timing of origin firing (Tanaka et al. 2011). Furthermore, simultaneous overexpression of several limiting replication factors advances late origin firing (Mantiero et al. 2011; Tanaka et al. 2011).Under genotoxic stress during S phase, DNA damage checkpoint pathways inhibit late origin firing (Zegerman and Diffley 2009). In budding yeast, DNA damage activates the mammalian ATM/ATR homolog, Mec1 kinase, which in turn activates the Rad53 effector kinase (the homolog of mammalian Chk2) to phosphorylate and inhibit the activities of Sld3 and Dbf4, thereby preventing late origin firing (Lopez-Mosqueda et al. 2010; Zegerman and Diffley 2010). Some firing of late origins could be detected under DNA damaging conditions in phosphorylation mutants of these two targets rendered refractory to the inhibition by Rad53. An initiation inhibitory activity within the nonstructured, amino-terminal regulatory domain of Mcm4 (Fig. 1) also plays a role in regulating origin firing under genotoxic stress (Sheu et al. 2014). Because this domain is a target of DDK (Masai et al. 2006; Sheu and Stillman 2006, 2010), it is conceivable that Mcm4 could mediate the checkpoint control by Rad53 phosphorylation of Dbf4. However, since DDK has targets other than Mcm4 and Mcm4 is regulated by signals in addition to DDK, a more comprehensive picture of how these factors cooperate to control origin firing under stress conditions remains to be addressed.Open in a separate windowFigure 1.Diagram of the Mcm4 subunit of Mcm2-7 helicase. The two overlapping segments within the Mcm4 structurally disordered N-terminal serine/threonine-rich domain (NSD) are shown.In addition to origin activation, DNA synthesis can be controlled at the level of replication fork progression. For example, deoxyribonucleoside triphosphate (dNTP) levels influence the rate of replication fork progression (Santocanale and Diffley 1998; Alvino et al. 2007). Hydroxyurea (HU) inhibits the activity of ribonucleotide reductase (RNR) and causes a dramatic slowdown of replication fork progression. In contrast, high dNTP concentration inhibits ORC-dependent initiation of DNA replication (Chabes and Stillman 2007). It has been proposed that dNTP levels are key determinants of replication fork speed, and cells adapt to replication stress by up-regulating dNTP pools (Poli et al. 2012). Methyl methanesulfonate (MMS), a DNA-alkylating agent, also results in slower fork progression while activating the DNA damage checkpoint response (Tercero and Diffley 2001). Although Mec1 and Rad53 are essential for preventing DNA replication fork catastrophe, these checkpoint kinases are not required for fork slowing in MMS (Tercero and Diffley 2001; Tercero et al. 2003). Thus, it is possible that an alternative mechanism might regulate fork progression under stress conditions.The structurally disordered N-terminal serine/threonine-rich domain (NSD) of Mcm4 participates in both initiation and fork progression (Sheu et al. 2014). It can be subdivided into two overlapping but functionally distinct segments, the proximal segment and the distal segment (Fig. 1). The proximal segment of the NSD (amino acids 74–174) is responsible for the initiation inhibitory activity that is mitigated by DDK through phosphorylation (Sheu and Stillman 2006, 2010). The distal segment (amino acids 2–145) is important for controlling fork progression and checkpoint response under replication stress caused by depletion of dNTP pools and its function is regulated by CDK (Devault et al. 2008; Sheu et al. 2014). Thus, this intrinsic regulatory domain of the replicative helicase may cooperate with additional factors to control origin firing and replication fork progression in response to various environmental conditions. Herein, we examine the contributions of Mcm4, Sld3, and Dbf4 in DNA damage–induced control of both origin activation and DNA replication fork progression.     

A unique DNA entry gate serves for regulated loading of the eukaryotic replicative helicase MCM2–7 onto DNA

The regulated loading of the replicative helicase minichromosome maintenance proteins 2–7 (MCM2–7) onto replication origins is a prerequisite for replication fork establishment and genomic stability. Origin recognition complex (ORC), Cdc6, and Cdt1 assemble two MCM2–7 hexamers into one double hexamer around dsDNA. Although the MCM2–7 hexamer can adopt a ring shape with a gap between Mcm2 and Mcm5, it is unknown which Mcm interface functions as the DNA entry gate during regulated helicase loading. Here, we establish that the Saccharomyces cerevisiae MCM2–7 hexamer assumes a closed ring structure, suggesting that helicase loading requires active ring opening. Using a chemical biology approach, we show that ORC–Cdc6–Cdt1-dependent helicase loading occurs through a unique DNA entry gate comprised of the Mcm2 and Mcm5 subunits. Controlled inhibition of DNA insertion triggers ATPase-driven complex disassembly in vitro, while in vivo analysis establishes that Mcm2/Mcm5 gate opening is essential for both helicase loading onto chromatin and cell cycle progression. Importantly, we demonstrate that the MCM2–7 helicase becomes loaded onto DNA as a single hexamer during ORC/Cdc6/Cdt1/MCM2–7 complex formation prior to MCM2–7 double hexamer formation. Our study establishes the existence of a unique DNA entry gate for regulated helicase loading, revealing key mechanisms in helicase loading, which has important implications for helicase activation.     

Characterization of a baculovirus lacking the DBP (DNA-binding protein) gene

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes two proteins that possess properties typical of single-stranded DNA-binding proteins (SSBs), late expression factor-3 (LEF-3), and a protein referred to as DNA-binding protein (DBP). Whereas LEF-3 is a multi-functional protein essential for viral DNA replication, transporting helicase into the nucleus, and forms a stable complex with the baculovirus alkaline nuclease, the role for DBP in baculovirus replication remains unclear. Therefore, to better understand the functional role of DBP in viral replication, a DBP knockout virus was generated from an AcMNPV bacmid and analyzed. The results of a growth curve analysis indicated that the dbp knockout construct was unable to produce budded virus indicating that dbp is essential. The lack of DBP does not cause a general shutdown of the expression of viral genes, as was revealed by accumulation of early (LEF-3), late (VP39), and very late (P10) proteins in cells transfected with the dbp knockout construct. To investigate the role of DBP in DNA replication, a real-time PCR-based assay was employed and showed that, although viral DNA synthesis occurred in cells transfected with the dbp knockout, the levels were less than that of the control virus suggesting that DBP is required for normal levels of DNA synthesis or for stability of nascent viral DNA. In addition, analysis of the viral DNA replicated by the dbp knockout by using field inversion gel electrophoresis failed to detect the presence of genome-length DNA. Furthermore, analysis of DBP from infected cells indicated that similar to LEF-3, DBP was tightly bound to viral chromatin. Assessment of the cellular localization of DBP relative to replicated viral DNA by immunoelectron microscopy indicated that, at 24 h post-infection, DBP co-localized with nascent DNA at distinct electron-dense regions within the nucleus. Finally, immunoelectron microscopic analysis of cells transfected with the dbp knockout revealed that DBP is required for the production of normal-appearing nucleocapsids and for the generation of the virogenic stroma.     

Different sources of “help” facilitate the antibody response to hepatitis D virus δ antigen

Repeated injections of hepatitis D antigen (HDAg) delivered either as a recombinant protein, or expressed from a DNA vaccine elicited no (or only very low) antibody responses in inbred mouse strains. Codelivery of oligonucleotides (ODN) with immune-stimulating sequences (ISS) with the protein antigen, or ISS in DNA vaccines (encoding HDAg) did not overcome the low intrinsic immunogenicity of this small viral antigen for B cells. In contrast, codelivery of immunogenic, heterologous proteins (either mixed to recombinant HDAg as recombinant proteins, or fused to HDAg sequences as chimeric antigens expressed from DNA vaccines) provided specific, CD4⁺ T cell-dependent help that supported efficient priming of antibody responses to HDAg. Chimeric proteins in which selected HDAg fragments were fused in frame with immunogenic, heterologous protein fragments produced by DNA vaccines allowed the mapping of antibody-binding HDAg domains of the viral antigen. The described approach thus facilitates induction of serum antibody responses against native viral antigens with low immunogenicity for B cells     

Dual role of tumor suppressor p53 in regulation of DNA replication and oncogene E6-promoter activity of epidermodysplasia verruciformis-associated human papillomavirus type 8

Human papillomavirus 8 (HPV8) is a representative of Epidermodysplasia verruciformis (EV)-associated viruses. Transient assays in the human skin keratinocyte cell line RTS3b have shown that its replication depends in trans on expression of the viral proteins E1 and E2, similarly to other HPVs. Using deletion mutants and cloned subfragments of the noncoding region (NCR) of HPV8 we identified a 65-bp sequence in the 3' part of the NCR to be necessary and sufficient to support replication in cis. The origin of replication (ori) of HPV8 is composed of the sequence motifs "CCAAC" (nt 57-73) and M29 (nt 84-112), which are highly conserved among the majority of EV HPVs. Analysis of M29 revealed an unconventional binding site of the E2 protein and an overlapping DNA recognition site of the tumor suppressor protein p53. Both these factors competitively bind to M29. In transient replication assays p53 acted as a potent inhibitor of ori activity, most probably in a DNA-binding-dependent fashion. The minimal ori sequences are also functionally critical for the E6 oncogene promoter P(175). In contrast to its effect on replication, p53 stimulated promoter activity depending on its interaction with M29. Our observations suggest that p53 is involved in controlling the balance between DNA replication and gene expression of HPV8.