Catalytic Properties of RNase BN/RNase Z from Escherichia coli: RNase BN IS BOTH AN EXO- AND ENDORIBONUCLEASE*

Processing of the 3′ terminus of tRNA in many organisms is carried out by an endoribonuclease termed RNase Z or 3′-tRNase, which cleaves after the discriminator nucleotide to allow addition of the universal -CCA sequence. In some eubacteria, such as Escherichia coli, the -CCA sequence is encoded in all known tRNA genes. Nevertheless, an RNase Z homologue (RNase BN) is still present, even though its action is not needed for tRNA maturation. To help identify which RNA molecules might be potential substrates for RNase BN, we carried out a detailed examination of its specificity and catalytic potential using a variety of synthetic substrates. We show here that RNase BN is active on both double- and single-stranded RNA but that duplex RNA is preferred. The enzyme displays a profound base specificity, showing no activity on runs of C residues. RNase BN is strongly inhibited by the presence of a 3′-CCA sequence or a 3′-phosphoryl group. Digestion by RNase BN leads to 3-mers as the limit products, but the rate slows on molecules shorter than 10 nucleotides in length. Most interestingly, RNase BN acts as a distributive exoribonuclease on some substrates, releasing mononucleotides and a ladder of digestion products. However, RNase BN also cleaves endonucleolytically, releasing 3′ fragments as short as 4 nucleotides. Although the presence of a 3′-phosphoryl group abolishes exoribonuclease action, it has no effect on the endoribonucleolytic cleavages. These data suggest that RNase BN may differ from other members of the RNase Z family, and they provide important information to be considered in identifying a physiological role for this enzyme.Maturation of tRNA precursors requires the removal of 5′ and 3′ precursor-specific sequences to generate the mature, functional tRNA (1). In eukaryotes, archaea, and certain eubacteria, the 3′-processing step is carried out by an endoribonuclease termed RNase Z or 3′-tRNase (2–6). However, in some bacteria, such as Escherichia coli, removal of 3′ extra residues is catalyzed by any of a number of exoribonucleases (7, 8). The major determinant for which mode of 3′-processing is utilized appears to be whether or not the universal 3′-terminal CCA sequence is encoded (2, 9). Thus, for those tRNA precursors in which the CCA sequence is absent, endonucleolytic cleavage by RNase Z right after the discriminator nucleotide generates a substrate for subsequent CCA addition by tRNA nucleotidyltransferase (1–3, 10). In view of this role for RNase Z in 3′-tRNA maturation, it is surprising that E. coli, an organism in which the CCA sequence is encoded in all tRNA genes (2), nevertheless contains an RNase Z homologue (11), because its action would appear not to be necessary. In fact, the physiological function of this enzyme in E. coli remains unclear, because mutants lacking this protein have no obvious growth phenotype (12). Hence, there is considerable interest in understanding the enzymatic capabilities of this enzyme.The E. coli RNase Z homologue initially was identified as a zinc phosphodiesterase (11) encoded by the elaC gene (now called rbn) (13). Subsequent work showed that the protein also displayed endoribonuclease activity on certain tRNA precursors in vitro (6, 14). However, more recent studies revealed that this protein actually is RNase BN, an enzyme originally discovered in 1983 and shown to be essential for maturation of those bacteriophage T4 tRNA precursors that lack a CCA sequence (15, 16). Using synthetic mimics of these T4 tRNA precursors, RNase BN was found to remove their 3′-terminal residue as a mononucleotide to generate a substrate for tRNA nucleotidyltransferase. Based on these reactions RNase BN was originally thought to be an exoribonuclease (13, 15, 17). However, subsequent work by us and others showed that it can act as an endoribonuclease on tRNA precursors (13, 18). RNase BN is required for maturation of tRNA precursors in E. coli mutant strains devoid of all other 3′-tRNA maturation exoribonucleases, although it is the least efficient RNase in this regard (7, 19). Thus, under normal circumstances, it is unlikely that RNase BN functions in maturation of tRNA in vivo except in phage T4-infected cells (15, 16).To obtain additional information on what types of RNA molecules might be substrates for RNase BN and to clarify whether it is an exo- or endoribonuclease, we have carried out a detailed examination of its catalytic properties and substrate specificity. We show here that RNase BN has both exo- and endoribonuclease activity and that it can act on a wide variety of RNA substrates. These findings suggest that E. coli RNase BN may differ from other members of the RNase Z family of enzymes.     

In Vitro Characterization of a Recombinant Blh Protein from an Uncultured Marine Bacterium as a ��-Carotene 15,15��-Dioxygenase

Codon optimization was used to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in Escherichia coli. The expressed enzyme cleaved β-carotene at its central double bond (15,15′) to yield two molecules of all-trans-retinal. The molecular mass of the native purified enzyme was ∼64 kDa as a dimer of 32-kDa subunits. The K_m, k_cat, and k_cat/K_m values for β-carotene as substrate were 37 μm, 3.6 min⁻¹, and 97 mm⁻¹ min⁻¹, respectively. The enzyme exhibited the highest activity for β-carotene, followed by β-cryptoxanthin, β-apo-4′-carotenal, α-carotene, and γ-carotene in decreasing order, but not for β-apo-8′-carotenal, β-apo-12′-carotenal, lutein, zeaxanthin, or lycopene, suggesting that the presence of one unsubstituted β-ionone ring in a substrate with a molecular weight greater than C₃₅ seems to be essential for enzyme activity. The oxygen atom of retinal originated not from water but from molecular oxygen, suggesting that the enzyme was a β-carotene 15,15′-dioxygenase. Although the Blh protein and β-carotene 15,15′-monooxygenases catalyzed the same biochemical reaction, the Blh protein was unrelated to the mammalian β-carotene 15,15′-monooxygenases as assessed by their different properties, including DNA and amino acid sequences, molecular weight, form of association, reaction mechanism, kinetic properties, and substrate specificity. This is the first report of in vitro characterization of a bacterial β-carotene-cleaving enzyme.Vitamin A (retinol) is a fat-soluble vitamin and important for human health. In vivo, the cleavage of β-carotene to retinal is an important step of vitamin A synthesis. The cleavage can proceed via two different biochemical pathways (1, 2). The major pathway is a central cleavage catalyzed by mammalian β-carotene 15,15′-monooxygenases (EC 1.14.99.36). β-Carotene is cleaved by the enzyme symmetrically into two molecules of all-trans-retinal, and retinal is then converted to vitamin A in vivo (3–5). The second pathway is an eccentric cleavage that occurs at double bonds other than the central 15,15′-double bond of β-carotene to produce β-apo-carotenals with different chain lengths, which are catalyzed by carotenoid oxygenases from mammals, plants, and cyanobacteria (6). These β-apo-carotenals are degraded to one molecule of retinal, which is subsequently converted to vitamin A in vivo (2).β-Carotene 15,15′-monooxygenase was first isolated as a cytosolic enzyme by identifying the product of β-carotene cleavage as retinal (7). The characterization of the enzyme and the reaction pathway from β-carotene to retinal were also investigated (4, 8). The enzyme activity has been found in mammalian intestinal mucosa, jejunum enterocytes, liver, lung, kidney, and brain (5, 9, 10). Molecular cloning, expression, and characterization of β-carotene 15,15′-monooxygenase have been reported from various species, including chickens (11), fruit flies (12), humans (13), mice (14), and zebra fishes (15).Other proteins thought to convert β-carotene to retinal include bacterioopsin-related protein (Brp) and bacteriorhodopsin-related protein-like homolog protein (Blh) (16). Brp protein is expressed from the bop gene cluster, which encodes the structural protein bacterioopsin, consisting of at least three genes as follows: bop (bacterioopsin), brp (bacteriorhodopsin-related protein), and bat (bacterioopsin activator) (17). brp genes were reported in Haloarcula marismortui (18), Halobacterium sp. NRC-1 (19), Halobacterium halobium (17), Haloquadratum walsbyi, and Salinibacter ruber (20). Blh protein is expressed from the proteorhodopsin gene cluster, which contains proteorhodopsin, crtE (geranylgeranyl-diphosphate synthase), crtI (phytoene dehydrogenase), crtB (phytoene synthase), crtY (lycopene cyclase), idi (isopentenyl diphosphate isomerase), and blh gene (21). Sources of blh genes were previously reported in Halobacterium sp. NRC-1 (19), Haloarcula marismortui (18), Halobacterium salinarum (22), uncultured marine bacterium 66A03 (16), and uncultured marine bacterium HF10 49E08 (21). β-Carotene biosynthetic genes crtE, crtB, crtI, crtY, ispA, and idi encode the enzymes necessary for the synthesis of β-carotene from isopentenyl diphosphate, and the Idi, IspA, CrtE, CrtB, CrtI, and CrtY proteins have been characterized in vitro (23–28). Blh protein has been proposed to catalyze or regulate the conversion of β-carotene to retinal (29, 30), but there is no direct proof of the enzymatic activity.In this study, we used codon optimization to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in Escherichia coli, and we performed a detailed biochemical and enzymological characterization of the expressed Blh protein. In addition, the properties of the enzyme were compared with those of mammalian β-carotene 15,15′-monooxygenases.     

Pif1 Helicase Lengthens Some Okazaki Fragment Flaps Necessitating Dna2 Nuclease/Helicase Action in the Two-nuclease Processing Pathway

We have developed a system to reconstitute all of the proposed steps of Okazaki fragment processing using purified yeast proteins and model substrates. DNA polymerase δ was shown to extend an upstream fragment to displace a downstream fragment into a flap. In most cases, the flap was removed by flap endonuclease 1 (FEN1), in a reaction required to remove initiator RNA in vivo. The nick left after flap removal could be sealed by DNA ligase I to complete fragment joining. An alternative pathway involving FEN1 and the nuclease/helicase Dna2 has been proposed for flaps that become long enough to bind replication protein A (RPA). RPA binding can inhibit FEN1, but Dna2 can shorten RPA-bound flaps so that RPA dissociates. Recent reconstitution results indicated that Pif1 helicase, a known component of fragment processing, accelerated flap displacement, allowing the inhibitory action of RPA. In results presented here, Pif1 promoted DNA polymerase δ to displace strands that achieve a length to bind RPA, but also to be Dna2 substrates. Significantly, RPA binding to long flaps inhibited the formation of the final ligation products in the reconstituted system without Dna2. However, Dna2 reversed that inhibition to restore efficient ligation. These results suggest that the two-nuclease pathway is employed in cells to process long flap intermediates promoted by Pif1.Eukaryotic cellular DNA is replicated semi-conservatively in the 5′ to 3′ direction. A leading strand is synthesized by DNA polymerase ϵ in a continuous manner in the direction of opening of the replication fork (1, 2). A lagging strand is synthesized by DNA polymerase δ (pol δ)³ in the opposite direction in a discontinuous manner, producing segments called Okazaki fragments (3). These stretches of ∼150 nucleotides (nt) must be joined together to create the continuous daughter strand. DNA polymerase α/primase (pol α) initiates each fragment by synthesizing an RNA/DNA primer consisting of ∼1-nt of RNA and ∼10–20 nt of DNA (4). The sliding clamp proliferating cell nuclear antigen (PCNA) is loaded on the DNA by replication factor C (RFC). pol δ then complexes with PCNA and extends the primer. When pol δ reaches the 5′-end of the downstream Okazaki fragment, it displaces the end into a flap while continuing synthesis, a process known as strand displacement (5, 6). These flap intermediates are cleaved by nucleases to produce a nick for DNA ligase I (LigI) to seal, completing the DNA strand.In one proposed mechanism for flap processing, the only required nuclease is flap endonuclease 1 (FEN1). pol δ displaces relatively short flaps, which are cleaved by FEN1 as they are created, leaving a nick for LigI (7–9). FEN1 binds at the 5′-end of the flap and tracks down the flap cleaving only at the base (5, 10, 11). Because pol δ favors the displacement of RNA-DNA hybrids over DNA-DNA hybrids, strand displacement generally is limited to that of the initiator RNA of an Okazaki fragment (12). In addition, the tightly coordinated action of pol δ and FEN1 also tends to keep flaps short. However, biochemical reconstitution studies demonstrate that some flaps can become long (13, 14). Once these flaps reach ∼30 nt, they can be bound by the eukaryotic single strand binding protein replication protein A (RPA) (15). Binding by RPA to a flap substrate inhibits cleavage by FEN1 (16). The RPA-bound flap would then require another mechanism for proper processing.This second mechanism is proposed to utilize Dna2 (16) in addition to FEN1. Dna2 is both a 5′-3′ helicase and an endonuclease (17, 18). Like FEN1, Dna2 recognizes 5′-flap structures, binding at the 5′-end of the flap and tracking downward toward the base (19, 20). Unlike FEN1, Dna2 cleaves the flap multiple times but not all the way to the base, such that a short flap remains (20). RPA binding to a flap has been shown to stimulate Dna2 cleavage (16). Therefore, if a flap becomes long enough to bind RPA, Dna2 binds and cleaves it to a length of 5–10 nucleotides from which RPA dissociates (21). FEN1 can then enter the flap, displace the Dna2, and then cleave at the base to make the nick for ligation (16, 18, 22). The need for this mechanism may be one reason why DNA2 is an essential gene in Saccharomyces cerevisiae (23, 24). It has been proposed that, in the absence of Dna2, flaps that become long enough to bind RPA cannot be properly processed, leading to genomic instability and cell death (23).In reconstitution of Okazaki fragment processing with purified proteins, even though some flaps became long enough to bind RPA, FEN1 was very effective at cleaving essentially all of the generated flaps (13, 14). Evidently, FEN1 could engage the flaps before binding of RPA. However, these reconstitution assays did not include the 5′-3′ helicase Pif1 (25, 26). Pif1 is involved in telomeric and mitochondrial DNA maintenance (26) and was first implicated in Okazaki fragment processing from genetic studies in S. cerevisiae. Deletion of PIF1 rescued the lethality of dna2Δ, although the double mutant was still temperature-sensitive (27). The authors of this report proposed that Pif1 creates a need for Dna2 by promoting longer flaps. Further supporting this conclusion, deletion of POL32, which encodes the subunit of pol δ that interacts with PCNA, rescued the temperature sensitivity of the dna2Δpif1Δ double mutant (12, 27). Importantly, pol δ exhibited reduced strand displacement activity when POL32 was deleted (12, 28, 29). The combination of pif1Δ and pol32Δ is believed to create a situation in which virtually no long flaps are formed, eliminating the requirement for Dna2 flap cleavage (27).We recently performed reconstitution assays showing that Pif1 can assist in the creation of long flaps. Inclusion of Pif1, in the absence of RPA, increased the proportion of flaps that lengthened to ∼28–32 nt before FEN1 cleavage (14). With the addition of RPA, the appearance of these long flap cleavage products was suppressed. Evidently, Pif1 promoted such rapid flap lengthening that RPA bound some flaps before FEN1 and inhibited cleavage. The RPA-bound flaps would presumably require cleavage by Dna2 for proper processing.Only a small fraction of flaps became long with Pif1. However, there are hundreds of thousands of Okazaki fragments processed per replication cycle (30). Therefore, thousands of flaps are expected to be lengthened by Pif1 in vivo, a number significant enough that improper processing of such flaps could lead to cell death.Our goal here was to determine whether Pif1 can influence the flow of Okazaki fragments through the two proposed pathways. We first questioned whether Pif1 stimulates strand displacement synthesis by pol δ. Next, we asked whether Pif1 lengthens short flaps so that Dna2 can bind and cleave. Finally, we used a complete reconstitution system to determine whether Pif1 promotes creation of RPA-bound flaps that require cleavage by both Dna2 and FEN1 before they can be ligated. Our results suggest that Pif1 promotes the two-nuclease pathway, and reveal the mechanisms involved.     

Mapping of the Functional Boundaries and Secondary Structure of the Mouse Mammary Tumor Virus Rem-responsive Element

Mouse mammary tumor virus (MMTV) is a complex retrovirus that encodes at least three regulatory and accessory proteins, including Rem. Rem is required for nuclear export of unspliced viral RNA and efficient expression of viral proteins. Our previous data indicated that sequences at the envelope-3′ long terminal repeat junction are required for proper export of viral RNA. To further map the Rem-responsive element (RmRE), reporter vectors containing various portions of the viral envelope gene and the 3′ long terminal repeat were tested in the presence and absence of Rem in transient transfection assays. A 476-bp fragment that spans the envelope-long terminal repeat junction had activity equivalent to the entire 3′-end of the mouse mammary tumor virus genome, but further deletions at the 5′- or 3′-ends reduced Rem responsiveness. RNase structure mapping of the full-length RmRE and a 3′-truncation suggested multiple domains with local base pairing and intervening single-stranded segments. A secondary structure model constrained by these data is reminiscent of the RNA response elements of other complex retroviruses, with numerous local stem-loops and long-range base pairs near the 5′- and 3′-boundaries, and differs substantially from an earlier model generated without experimental constraints. Covariation analysis provides limited support for basic features of our model. Reporter assays in human and mouse cell lines revealed similar boundaries, suggesting that the RmRE does not require cell type-specific proteins to form a functional structure.Mouse mammary tumor virus (MMTV)³ has multiple regulatory and accessory genes (1, 2). The known accessory genes specify a dUTPase (3), which is believed to be involved in retroviral replication in non-dividing cells (4), as well as superantigen (Sag). Sag is a transmembrane glycoprotein that is involved in the lymphocyte-mediated transmission of MMTV from maternal milk in the gut to susceptible epithelial cells in the mammary gland (5, 6). The Sag protein expressed by endogenous (germline) MMTV proviruses has been reported to provide susceptibility to infection by exogenous MMTVs or the bacterial pathogen, Vibrio cholerae (7). These results suggest a role for MMTV Sag in the host innate immune response.MMTV recently was shown to be a complex retrovirus (1). Complex retroviruses encode RNA-binding proteins that facilitate nuclear export of unspliced viral RNA by using a leucine-rich nuclear export sequence (8), which binds to chromosome region maintenance 1 (Crm1)(9), whereas simple retroviruses have a cis-acting constitutive transport element that directly interacts with components of the Tap/NXF1 pathway (10). Similar to other complex retroviruses, MMTV encodes a Rev-like protein, regulator of export/expression of MMTV mRNA (Rem) (1). Rem is translated from a doubly spliced mRNA into a 33-kDa protein that contains nuclear and nucleolar localization signals as well as a predicted RNA-binding motif and leucine-rich nuclear export sequence (1, 2). Our previous experiments indicated that Rem affects export of unspliced viral RNA, and a reporter vector that relies on luciferase expression from unspliced RNAs has increased activity in the presence of Rem (1). Sequences at the MMTV envelope-long terminal repeat (LTR) junction were required within the vector for Rem-induced expression, suggesting that the LTR contains all or part of the Rem-responsive element (RmRE). Very recently, Müllner et al. (11) identified a 490-nt region spanning the MMTV envelope-3′ LTR region, which was predicted to form a highly structured RNA element. This element confers Rem responsiveness on heterologous human immunodeficiency virus type 1 (HIV-1)-based plasmid constructs in transfection experiments.Experiments using other retroviral export proteins have demonstrated considerable variation in the size of the response elements. A minimal Rev-responsive element (RRE) in the human immunodeficiency virus type 1 (HIV-1) genomic RNA is 234 nt, the human T-cell leukemia virus Rex-responsive element is 205 nt (12–14), whereas the Rec-responsive element (RcRE; also known as the K-RRE) of human endogenous retrovirus type K is 416 to 429 nt (15, 16). Most response elements are confined to the 3′-end of their respective retroviral genomes (either to the envelope or LTR regions) (14, 15), but 5′ Rev-response elements also have been identified (17). Studies indicate that the secondary structure is a critical factor for proper function of retroviral response elements (18), and that multiple stem-loops are required. Export proteins multimerize on these elements to allow activity (19).In the current study, we have used deletion mutations within a reporter vector based on the 3′-end of the MMTV genome to define a 476-nt element necessary for maximum Rem responsiveness. This element spans the envelope-LTR junction of the MMTV genome as previously reported (1). However, a secondary structure model generated using digestions of the RmRE by RNases V1, T1, and A as experimental constraints differs significantly from the published structure (11) and more closely resembles complex retroviral response elements. Transfection experiments indicated that the MMTV RmRE could function in both mouse and human cells, suggesting that conserved cellular proteins interact with Rem.     

Repeated Domains of Leptospira Immunoglobulin-like Proteins Interact with Elastin and Tropoelastin

Leptospira spp., the causative agents of leptospirosis, adhere to components of the extracellular matrix, a pivotal role for colonization of host tissues during infection. Previously, we and others have shown that Leptospira immunoglobulin-like proteins (Lig) of Leptospira spp. bind to fibronectin, laminin, collagen, and fibrinogen. In this study, we report that Leptospira can be immobilized by human tropoelastin (HTE) or elastin from different tissues, including lung, skin, and blood vessels, and that Lig proteins can bind to HTE or elastin. Moreover, both elastin and HTE bind to the same LigB immunoglobulin-like domains, including LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12 as demonstrated by enzyme-linked immunosorbent assay (ELISA) and competition ELISAs. The LigB immunoglobulin-like domain binds to the 17th to 27th exons of HTE (17–27HTE) as determined by ELISA (LigBCon4, K_D = 0.50 μm; LigBCen7′–8, K_D = 0.82 μm; LigBCen9, K_D = 1.54 μm; and LigBCen12, K_D = 0.73 μm). The interaction of LigBCon4 and 17–27HTE was further confirmed by steady state fluorescence spectroscopy (K_D = 0.49 μm) and ITC (K_D = 0.54 μm). Furthermore, the binding was enthalpy-driven and affected by environmental pH, indicating it is a charge-charge interaction. The binding affinity of LigBCon4D341N to 17–27HTE was 4.6-fold less than that of wild type LigBCon4. In summary, we show that Lig proteins of Leptospira spp. interact with elastin and HTE, and we conclude this interaction may contribute to Leptospira adhesion to host tissues during infection.Pathogenic Leptospira spp. are spirochetes that cause leptospirosis, a serious infectious disease of people and animals (1, 2). Weil syndrome, the severe form of leptospiral infection, leads to multiorgan damage, including liver failure (jaundice), renal failure (nephritis), pulmonary hemorrhage, meningitis, abortion, and uveitis (3, 4). Furthermore, this disease is not only prevalent in many developing countries, it is reemerging in the United States (3). Although leptospirosis is a serious worldwide zoonotic disease, the pathogenic mechanisms of Leptospira infection remain enigmatic. Recent breakthroughs in applying genetic tools to Leptospira may facilitate studies on the molecular pathogenesis of leptospirosis (5–8).The attachment of pathogenic Leptospira spp. to host tissues is critical in the early phase of Leptospira infection. Leptospira spp. adhere to host tissues to overcome mechanical defense systems at tissue surfaces and to initiate colonization of specific tissues, such as the lung, kidney, and liver. Leptospira invade hosts tissues through mucous membranes or injured epidermis, coming in contact with subepithelial tissues. Here, certain bacterial outer surface proteins serve as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs)² to mediate the binding of bacteria to different extracellular matrices (ECMs) of host cells (9). Several leptospiral MSCRAMMs have been identified (10–18), and we speculate that more will be identified in the near future.Lig proteins are distributed on the outer surface of pathogenic Leptospira, and the expression of Lig protein is only found in low passage strains (14, 16, 17), probably induced by environmental cues such as osmotic or temperature changes (19). Lig proteins can bind to fibrinogen and a variety of ECMs, including fibronectin (Fn), laminin, and collagen, thereby mediating adhesion to host cells (20–23). Lig proteins also constitute good vaccine candidates (24–26).Elastin is a component of ECM critical to tissue elasticity and resilience and is abundant in skin, lung, blood vessels, placenta, uterus, and other tissues (27–29). Tropoelastin is the soluble precursor of elastin (28). During the major phase of elastogenesis, multiple tropoelastin molecules associate through coacervation (30–32). Because of the abundance of elastin or tropoelastin on the surface of host cells, several bacterial MSCRAMMs use elastin and/or tropoelastin to mediate adhesion during the infection process (33–35).Because leptospiral infection is known to cause severe pulmonary hemorrhage (36, 37) and abortion (38), we hypothesize that some leptospiral MSCRAMMs may interact with elastin and/or tropoelastin in these elastin-rich tissues. This is the first report that Lig proteins of Leptospira interact with elastin and tropoelastin, and the interactions are mediated by several specific immunoglobulin-like domains of Lig proteins, including LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12, which bind to the 17th to 27th exons of human tropoelastin (HTE).     

Reconstitution of Uracil DNA Glycosylase-initiated Base Excision Repair in Herpes Simplex Virus-1

Herpes simplex virus-1 is a large double-stranded DNA virus that is self-sufficient in a number of genome transactions. Hence, the virus encodes its own DNA replication apparatus and is capable of mediating recombination reactions. We recently reported that the catalytic subunit of the HSV-1 DNA polymerase (UL30) exhibits apurinic/apyrimidinic and 5′-deoxyribose phosphate lyase activities that are integral to base excision repair. Base excision repair is required to maintain genome stability as a means to counter the accumulation of unusual bases and to protect from the loss of DNA bases. Here we have reconstituted a system with purified HSV-1 and human proteins that perform all the steps of uracil DNA glycosylase-initiated base excision repair. In this system nucleotide incorporation is dependent on the HSV-1 uracil DNA glycosylase (UL2), human AP endonuclease, and the HSV-1 DNA polymerase. Completion of base excision repair can be mediated by T4 DNA ligase as well as human DNA ligase I or ligase IIIα-XRCC1 complex. Of these, ligase IIIα-XRCC1 is the most efficient. Moreover, ligase IIIα-XRCC1 confers specificity onto the reaction in as much as it allows ligation to occur in the presence of the HSV-1 DNA polymerase processivity factor (UL42) and prevents base excision repair from occurring with heterologous DNA polymerases. Completion of base excision repair in this system is also dependent on the incorporation of the correct nucleotide. These findings demonstrate that the HSV-1 proteins in combination with cellular factors that are not encoded by the virus are capable of performing base excision repair. These results have implications on the role of base excision repair in viral genome maintenance during lytic replication and reactivation from latency.Herpes simplex virus-1 (HSV-1)² is a large double-stranded DNA virus with a genome of ∼152 kilobase pairs (for reviews, see Refs. 1 and 2). HSV-1 switches between lytic replication in epithelial cells and a state of latency in sensory neurons during which there is no detectable DNA replication (1). Viral DNA replication is mediated by seven essential virus-encoded factors (3–5). Of these, two encode subunits of the viral replicase (for review, see Refs. 6 and 7). The catalytic subunit (UL30) exhibits DNA polymerase (Pol), 3′-5′ proofreading exonuclease, and RNase H activities (8–11). UL30 exists as a heterodimer with the UL42 protein that confers a high degree of processivity on the Pol (11–17).Viral DNA replication is accompanied by vigorous recombination that leads to the formation of large networks of viral DNA replication intermediates (18). The HSV-1 single-strand DNA-binding protein (ICP8) has been shown to play a major role in mediating these recombination reactions (19–21). One role for the high frequency of recombination is to restart DNA replication at sites of fork collapse. Further mechanisms that contribute to genome maintenance are processes that survey and repair damage to the DNA to ensure the availability of a robust replication template. In this regard base excision repair (BER) is essential to remove unusual bases from the DNA and to repair apurinic/apyrimidinic (AP) sites resulting from spontaneous base loss (for review, see Ref. 22). With respect to HSV-1, a recent study showed that viral DNA from infected cultured fibroblasts contains a steady state of 2.8–5.9 AP sites per viral genome equivalent (23). Because AP sites are non-instructional, the failure to repair such sites would terminate viral replication. Indeed, UL30 cannot replicate beyond a model AP site (tetrahydrofuran residue) (23), indicating that the virus must enable a process to repair such lesions. In this regard HSV-1 possesses several enzymes that would safeguard from the accumulation of unusual bases, specifically uracil, and base loss. Hence, HSV-1 encodes a uracil DNA glycosylase (UDG) (UL2) as well as a dUTPase to reduce the pool of dUTP and prevent misincorporation by the viral Pol (24, 25). Moreover, we recently showed that the catalytic subunit of the viral Pol (UL30) exhibits AP and 5′-deoxyribose phosphate (dRP) lyase activities (26). The presence of a virus-encoded UDG and DNA lyase indicates that HSV-1 has the capacity to perform integral steps of BER, specifically for the removal of uracil. Indeed, the excision of uracil may be important for viral replication. Hence, it has been shown that uracil substitutions in the viral origins of replication alters their recognition by the viral initiator protein (27). Moreover, whereas UL2 may be dispensable for viral replication in fibroblast (24), UL2 mutants exhibit reduced neurovirulence and a decreased frequency of reactivation from latency (28). Thus, UDG action in HSV-1 may be important for viral reactivation after quiescence in neuronal cells during which the genome may accumulate uracil as a result of spontaneous deamination of cytosine. In another herpesvirus, cytomegalovirus, the viral UDG was shown to be required for the transition to late-phase DNA replication (29, 30). Consequently, it is possible that BER plays a significant role in various aspects of the herpesvirus life cycle.In mammalian single-nucleotide BER initiated by monofunctional DNA glycosylases, the resulting AP sites are incised hydrolytically at the 5′ side by AP endonuclease (APE), generating a 3′-OH. This is followed by template-directed incorporation of one nucleotide by Pol β to generate a 5′-dRP flap (22, 31, 32). The 5′-dRP residue is subsequently removed by the 5′-dRP lyase activity of Pol β to leave a nick with a 3′-OH and 5′-phosphate that is ligated by DNA ligase I or the physiologically more relevant ligase IIIα-XRCC1 complex (for review, see Refs. 33 and 34). Here we show that the HSV-1 UDG (UL2) and Pol (UL30) cooperate with human APE and human ligase IIIα-XRCC1 complex to perform BER in vitro. This finding has implications on the role of BER in viral genome maintenance during lytic replication and in the emergence of the virus from neuronal latency.     

The 3��-Flap Pocket of Human Flap Endonuclease 1 Is Critical for Substrate Binding and Catalysis

Flap endonuclease 1 (FEN1) proteins, which are present in all kingdoms of life, catalyze the sequence-independent hydrolysis of the bifurcated nucleic acid intermediates formed during DNA replication and repair. How FEN1s have evolved to preferentially cleave flap structures is of great interest especially in light of studies wherein mice carrying a catalytically deficient FEN1 were predisposed to cancer. Structural studies of FEN1s from phage to human have shown that, although they share similar folds, the FEN1s of higher organisms contain a 3′-extrahelical nucleotide (3′-flap) binding pocket. When presented with 5′-flap substrates having a 3′-flap, archaeal and eukaryotic FEN1s display enhanced reaction rates and cleavage site specificity. To investigate the role of this interaction, a kinetic study of human FEN1 (hFEN1) employing well defined DNA substrates was conducted. The presence of a 3′-flap on substrates reduced K_m and increased multiple- and single turnover rates of endonucleolytic hydrolysis at near physiological salt concentrations. Exonucleolytic and fork-gap-endonucleolytic reactions were also stimulated by the presence of a 3′-flap, and the absence of a 3′-flap from a 5′-flap substrate was more detrimental to hFEN1 activity than removal of the 5′-flap or introduction of a hairpin into the 5′-flap structure. hFEN1 reactions were predominantly rate-limited by product release regardless of the presence or absence of a 3′-flap. Furthermore, the identity of the stable enzyme product species was deduced from inhibition studies to be the 5′-phosphorylated product. Together the results indicate that the presence of a 3′-flap is the critical feature for efficient hFEN1 substrate recognition and catalysis.In eukaryotic DNA replication and repair, various bifurcated nucleic acid structure intermediates are formed and must be processed by the appropriate nuclease. Two examples of biological processes that create bifurcated DNA intermediates are Okazaki fragment maturation (1, 2) and long patch excision repair (3). In both models, a polymerase executes strand-displacement synthesis to create a double-stranded DNA (dsDNA)⁶ two-way junction from which a 5′-flap structure protrudes. The penultimate step of both pathways is the cleavage of this flap structure to create a nicked DNA that is then ligated. Because the bifurcated DNA structures that are formed in the aforementioned processes can theoretically occur anywhere in the genome, the nuclease associated with the cleavage of 5′-flap structures in eukaryotic cells, which is called flap endonuclease 1 (FEN1), must be capable of cleavage regardless of sequence. Therefore, FEN1 nucleases, which are found in all kingdoms of life (4), have evolved to recognize substrates based upon nucleic acid structure and strand polarity (5, 6).The Okazaki fragment maturation pathway of yeast has become a paradigm of eukaryotic lagging strand DNA synthesis. In the yeast model, bifurcated intermediates with large single-stranded DNA (ssDNA) 5′-flap structures are imprecisely cleaved by DNA2 in a replication protein A -dependent manner (7). Subsequent to the DNA2 cleavage, Rad27 (yeast homologue of FEN1) cleaves precisely to generate an intermediate suitable for ligation (2). The recent discovery that human DNA2 is predominantly located in mitochondria in various human cell lines (8, 9) suggests that hFEN1 is the paramount 5′-flap endonuclease in the nuclei of human cells. This observation potentially provides a plausible rationale for why deletion of RAD27 (yeast FEN1 homologue) is tolerated in Saccharomyces cerevisiae (10), whereas deletion of FEN1 in mammals is embryonically lethal (11). Recent models wherein mice carrying a mutation (E160D) in the FEN1 gene, which was shown in vitro to alter enzymatic properties (12), have demonstrated that FEN1 functional deficiency in mice (S129 and Black 6) increases the incidence of cancer, albeit different types presumably due to genetic background (13, 14). Thus, the function of mammalian FEN1 in vivo is vital to the prevention of genomic instability. In addition to its importance in the nucleus, hFEN1 has recently been detected in mitochondrial extracts (15, 16) and implicated in mitochondrial long patch base excision repair (15). Considering the pivotal roles of hFEN1 in DNA replication and repair, it is of interest to understand how hFEN1 and homologues achieve substrate and scissile phosphate selectivity in the absence of sequence information.Since its initial discovery as a nuclease that completes reconstituted Okazaki fragment maturation (17) and subsequent rediscovery as a 5′-flap-specific nuclease (DNaseIV) from bacteria (18), mouse (19), and HeLa cells (20), FEN1 proteins ranging from phage to human have been studied biochemically, computationally, and structurally (5, 6, 21). Biochemical characterizations of FEN1 proteins from various organisms have shown that this family of nucleases can perform phosphodiesterase activity on a wide variety of substrates; however, the efficiency of catalysis on various substrates differs among the species. For instance, phage FEN1s prefer pseudo-Y substrates (22, 23), whereas the archaeal and eukaryotic FEN1s prefer 5′-flap substrates (21, 24, 25), which have two dsDNA domains, one upstream and downstream of the site of cleavage, and a 5′-ssDNA protrusion (Fig. 1A). Primary sequence analysis indicates that FEN1 proteins share characteristic N-terminal (N) and Intermediate (I) “domains,” which harbor the highly conserved carboxylate residues that bind the requisite divalent metal ions (26–28). Structural studies of FEN1 nucleases from phage to humans (22, 29–36), have shown that the N and I domains comprise a single nuclease core domain consisting of a mixed, six- or seven-stranded β-sheet packed against an α-helical structure on both sides. The α-helices on either side of the β-sheet are “bridged” by a helical arch that spans the active site groove (supplemental Fig. S1). On one side of the β-sheet, the α-helical bundle (αb1) creates the floor of the active site and a DNA binding motif (helix-3-turn-helix) (32). Similarly, the opposite α-helical bundle (αb2) has also been observed to interact with DNA (35). Based on site-directed mutagenesis studies with T5 phage FEN1 (T5FEN1) (37) and hFEN1 (38, 39), and crystallographic studies of T4 phage FEN1 (T4FEN1) (22) and Archaeoglobus fulgidus FEN1 (aFEN1) (35) in complex with DNA, a general model for how FEN1 proteins recognize flap DNA has emerged. The helix-3-turn-helix motif is involved in downstream dsDNA binding, whereas the upstream dsDNA domain is bound by αb2. The helical arch is likely involved in 5′-flap binding (22).Open in a separate windowFIGURE 1.Secondary structure schematics of hFEN1 substrates. A, illustration of a general flap substrate created using a bimolecular approach whereby a template strand (T-strand), which partially folds into a hairpin, anneals with the duplex strand (d-strand). The T-strand hairpin creates the upstream dsDNA domain, whereas the d-strand base pairs with the T-strand to create the downstream dsDNA domain. The flap or any other structure is created by addition of nucleotides to the 5′-end of the d-strand. The interface between the upstream and downstream dsDNA domains may be viewed as a derivative of a two-way junction (74). Annealing of either the F(5), E, or G(15) d-strands with the T3F T-strand results in the formation of a (B) double flap substrate (Flap of 5-nt d-strand paired with a Template with a 3′-Flap, F(5)·T3F), C, exonuclease substrate with a 3′-extrahelical nucleotide (EXO d-strand paired with a Template with a 3′-Flap, E·T3F), and a D, fork-GEN substrate with a 3′-extrahelical nucleotide and a 15-nt ssDNA gap capped by a 23-nt hairpin structure (fork-Gap of 15-nt d-strand paired with a Template with a 3′-Flap, G(15)·T3F). E, annealing the F(5) d-strand with the T oligonucleotide creates a single flap (Flap of 5-nt d-strand paired with a Template, F(5)·T).Unlike phage FEN1s, studies of FEN1s from eubacterial (40), archaeal (21), and eukaryotic origins (41) have shown that the addition of a 3′-extrahelical nucleotide (3′-flap) to the upstream duplex of a 5′-flap substrate results in a rate enhancement and an increase in cleavage site specificity. Moreover, substrates possessing a 3′-flap, which mimic physiological “equilibrating flaps,” were cleaved exactly one nucleotide into the downstream duplex, thereby resulting in 5′-phosphorylated dsDNA product that was a suitable substrate for DNA ligase I (21, 41). As postulated by Kaiser et al. (21), the structure of an archaeal FEN1 in complex with dsDNA with a 3′-overhang showed that the protein contains a cleft adjacent to the upstream dsDNA binding site that binds the 3′-flap by means of van der Waals and hydrogen bonding interactions with the sugar moiety (35). Once the residues associated with 3′-flap binding were identified, sequence alignment analyses showed that the amino acid residues in the 3′-flap binding pocket are highly conserved from archaea to human. Furthermore, mutation of the conserved amino acid residues in the 3′-flap binding pocket of hFEN1 resulted in reduced affinity for and cleavage specificity on double flap substrates (42). Although the effects of the addition of a 3′-flap to substrates on hFEN1 catalysis are known qualitatively, a detailed understanding of the relationship between changes in catalytic parameters and rate enhancement by the presence of a 3′-flap is unknown. Here, we describe a detailed kinetic analysis of hFEN1 using four well characterized DNA substrates and show that the presence of a 3′-flap on a substrate not only contributes to substrate binding (42), but also increases multiple and single turnover rates of reaction in the presence of near physiological monovalent salt concentrations. We also demonstrate that, like T5FEN1, hFEN1 is rate-limited by product release, and thus multiple turnover rates at saturating concentrations of substrate are predominantly a reflection of product release and not catalysis as was previously concluded (39). Furthermore, this study provides insight into the mechanism of hFEN1 substrate recognition.