Putative Helicase Domain Research Articles

NAT10 resolves harmful nucleolar R-loops depending on its helicase domain and acetylation of DDX21

BackgroundAberrant accumulation of R-loops leads to DNA damage, genome instability and even cell death. Therefore, the timely removal of harmful R-loops is essential for the maintenance of genome integrity. Nucleolar R-loops occupy up to 50% of cellular R-loops due to the frequent activation of Pol I transcription. However, the mechanisms involved in the nucleolar R-loop resolution remain elusive. The nucleolar acetyltransferase NAT10 harbors a putative RecD helicase domain (RHD), however, if NAT10 acts in the R-loop resolution is still unknown.MethodsNAT10 knockdown cell lines were constructed using CRISPR/Cas9 technology and short hairpin RNA targeting NAT10 mRNA, respectively. The level of R-loops was detected by immunofluorescent staining combined with RNase H treatment. The helicase activity of NAT10 or DDX21 was determined by in vitro helicase experiment. The interaction between NAT10 and DDX21 was verified by co-immunoprecipitation, immunofluorescent staining and GST pull-down experiments. Acetylation sites of DDX21 by NAT10 were analyzed by mass spectrometry. NAT10 knockdown-induced DNA damage was evaluated by immunofluorescent staining and Western blot detecting γH2AX.ResultsDepletion of NAT10 led to the accumulation of nucleolar R-loops. NAT10 resolves R-loops through an RHD in vitro and in cells. However, Flag-NAT10 ∆RHD mutant still partially reduced R-loop levels in the NAT10-depleted cells, suggesting that NAT10 might resolve R-loops through additional pathways. Further, the acetyltransferase activity of NAT10 is required for the nucleolar R-loop resolution. NAT10 acetylates DDX21 at K236 and K573 to enhance the helicase activity of DDX21 to unwind nucleolar R-loops. The helicase activity of DDX21 significantly decreased by Flag-DDX21 2KR and increased by Flag-DDX21 2KQ in cells and in vitro. Consequently, NAT10 depletion-induced nucleolar R-loop accumulation led to DNA damage, which was rescued by co-expression of Flag-DDX21 2KQ and Flag-NAT10 G641E, demonstrating that NAT10 resolves nucleolar R-loops through bipartite pathways.ConclusionWe demonstrate that NAT10 is a novel R-loop resolvase and it resolves nucleolar R-loops depending on its helicase activity and acetylation of DDX21. The cooperation of NAT10 and DDX21 provides comprehensive insights into the nucleolar R-loop resolution for maintaining genome stability.

The Significance of the DUF283 Domain for the Activity of Human Ribonuclease Dicer.

Dicers are multidomain proteins, usually comprising an amino-terminal putative helicase domain, a DUF283 domain (domain of unknown function), a PAZ domain, two RNase III domains (RNase IIIa and RNase IIIb) and a dsRNA-binding domain. Dicer homologs play an important role in the biogenesis of small regulatory RNAs by cleaving single-stranded precursors adopting stem-loop structures (pre-miRNAs) and double-strand RNAs into short RNA duplexes containing functional microRNAs or small interfering RNAs, respectively. Growing evidence shows that apart from the canonical role, Dicer proteins can serve a number of other functions. For example, results of our previous studies showed that human Dicer (hDicer), presumably through its DUF283 domain, can facilitate hybridization between two complementary RNAs, thus, acting as a nucleic acid annealer. Here, to test this assumption, we prepared a hDicer deletion variant lacking the amino acid residues 625-752 corresponding to the DUF283 domain. The respective 128-amino acid fragment of hDicer was earlier demonstrated to accelerate base-pairing between two complementary RNAs in vitro. We show that the ΔDUF(625-752) hDicer variant loses the potential to facilitate RNA-RNA base pairing, which strongly proves our hypothesis about the importance of the DUF283 domain for the RNA-RNA annealing activity of hDicer. Interestingly, the in vitro biochemical characterization of the obtained deletion variant reveals that it displays different RNA cleavage properties depending on the pre-miRNA substrate.

Unknown Areas of Activity of Human Ribonuclease Dicer: A Putative Deoxyribonuclease Activity.

The Dicer ribonuclease plays a crucial role in the biogenesis of small regulatory RNAs (srRNAs) by processing long double-stranded RNAs and single-stranded hairpin RNA precursors into small interfering RNAs (siRNAs) and microRNAs (miRNAs), respectively. Dicer-generated srRNAs can control gene expression by targeting complementary transcripts and repressing their translation or inducing their cleavage. Human Dicer (hDicer) is a multidomain enzyme comprising a putative helicase domain, a DUF283 domain, platform, a PAZ domain, a connector helix, two RNase III domains (RNase IIIa and RNase IIIb) and a dsRNA-binding domain. Specific, ~20-base pair siRNA or miRNA duplexes with 2 nucleotide (nt) 3’-overhangs are generated by Dicer when an RNA substrate is anchored within the platform-PAZ-connector helix (PPC) region. However, increasing number of reports indicate that in the absence of the PAZ domain, binding of RNA substrates can occur by other Dicer domains. Interestingly, truncated variants of Dicer, lacking the PPC region, have been found to display a DNase activity. Inspired by these findings, we investigated how the lack of the PAZ domain, or the entire PPC region, would influence the cleavage activity of hDicer. Using immunopurified 3xFlag-hDicer produced in human cells and its two variants: one lacking the PAZ domain, and the other lacking the entire PPC region, we show that the PAZ domain deletion variants of hDicer are not able to process a pre-miRNA substrate, a dsRNA with 2-nt 3ʹ-overhangs, and a blunt-ended dsRNA. However, the PAZ deletion variants exhibit both RNase and DNase activity on short single-stranded RNA and DNAs, respectively. Collectively, our results indicate that when the PAZ domain is absent, other hDicer domains may contribute to substrate binding and in this case, non-canonical products can be generated.

Characterization and detection of maize-associated pteridovirus (MaPV), infecting maize (Zea mays) in the Arusha region of Tanzania

Maize lethal necrosis disease (MLND) is currently threatening maize production in a large area of East Africa. Synergistic infections of Maize chlorotic mottle virus (MCMV) and members of the Potyviridae family are known to elicit MLND. Metaviromics studies of plant viruses have allowed for the discovery of novel viral diversity, with a number of these viruses having recently been shown to co-occur with MCMV and Potyviruses. However, their contribution to the expression of disease symptoms still requires extensive research. In a survey for viruses associated with maize in Tanzania, 35 samples were sequenced using an RNA-tag-seq metaviromics approach. Bioinformatic analysis of assembled reads yielded two contigs (5.8 and 2.7 kbp) that shared sequence homology with RNA1 and RNA2 of the dsRNA Japanese holly fern mottle virus (JHFMoV) (genus: Pteridovirus) from a single sample collected in the Arusha region. RNA1 encodes for a polyprotein product containing putative viral methyltransferase, helicase and polymerase domains. RNA2 contains three open reading frames (ORFs), one of which encodes for a putative movement protein, while the remaining two encode for putative products with no known function. The tentative name Maize-associated pteridovirus (MaPV) has been proposed for the virus in this study.

Full-Genome Sequence of Porcine Circovirus type 3 recovered from serum of sows with stillbirths in Brazil.

Two full-genome sequences of porcine circovirus type 3 (PCV3) are reported. The genomes were recovered from pooled serum samples from sows who had just delivered litters with variable numbers of stillbirths. The two circular genomes (PCV3-BR/RS/6 and PCV3-BR/RS/8) are 2,000 nucleotides long and contain two open reading frames (ORFs) oriented in opposite directions that encode the putative capsid (Cap) and replicase (Rep) proteins. The intergenic region contains a stem-loop motif, as reported for other circoviruses. Rolling circle replication motifs and putative helicase domains were identified in the Rep coding region. The degree of overall nucleotide similarity between the genomes reported here and those available at GenBank was higher than 97%. No PCV3 sequence was detected in pooled serum samples from sows which had no stillbirths on the same farms. However, further studies are necessary to confirm the association between PCV3 and the occurrence of stillbirths.

Identification of Cleavage Sites Recognized by the 3C-Like Cysteine Protease within the Two Polyproteins of Strawberry Mottle Virus.

Strawberry mottle virus (SMoV, family Secoviridae, order Picornavirales) is one of several viruses found in association with strawberry decline disease in Eastern Canada. The SMoV genome consists of two positive-sense single-stranded RNAs, each encoding one large polyprotein. The RNA1 polyprotein (P1) includes the domains for a putative helicase, a VPg, a 3C-like cysteine protease and an RNA-dependent RNA polymerase at its C-terminus, and one or two protein domains at its N-terminus. The RNA2 polyprotein (P2) is predicted to contain the domains for a movement protein (MP) and one or several coat proteins at its N-terminus, and one or more additional domains for proteins of unknown function at its C-terminus. The RNA1-encoded 3C-like protease is presumed to cleave the two polyproteins in cis (P1) and in trans (P2). Using in vitro processing assays, we systematically scanned the two polyproteins for cleavage sites recognized by this protease. We identified five cis-cleavage sites in P1, with cleavage between the putative helicase and VPg domains being the most efficient. The presence of six protein domains in the SMoV P1, including two upstream of the putative helicase domain, is a feature shared with nepoviruses but not with comoviruses. Results from trans-cleavage assays indicate that the RNA1-encoded 3C-like protease recognized a single cleavage site, which was between the predicted MP and coat protein domains in the P2 polyprotein. The cleavage site consensus sequence for the SMoV 3C-like protease is AxE (E or Q)/(G or S).

Molecular characterization of a novel endornavirus from the phytopathogenic fungus Botrytis cinerea

The complete sequence of a novel endornavirus (Botrytis cinerea endornavirus 1, BcEV1) from the phytopathogenic fungus Botrytis cinerea strain HBtom-372 was determined. The BcEV1 coding strand is 11,557 nucleotides long, possessing an open reading frame (ORF) that codes for a polyprotein of 3,787 amino acid residues and lacks a site-specific nick. The polyprotein contains viral methyltransferase (MTR) domain, a cysteine-rich region (CRR), two putative viral helicase (DEXDc-like and Hel-1) domains, and an RNA-dependent RNA polymerase_2 (RdRp_2) domain. In phylogenetic analysis, BcEV1 clustered with several fungal endornaviruses, forming an independent clade, and it was detected in 4.2% of B. cinerea strains collected from central China.

Identification and characterization of RNA duplex unwinding and ATPase activities of an alphatetravirus superfamily 1 helicase

Dendrolimus punctatus tetravirus (DpTV) belongs to the genus omegatetravirus of the Alphatetraviridae family. Sequence analysis predicts that DpTV replicase contains a putative helicase domain (Hel). However, the helicase activity in alphatetraviruses has never been formally determined. In this study, we determined that DpTV Hel is a functional RNA helicase belonging to superfamily-1 helicase with 5′–3′ dsRNA unwinding directionality. Further characterization determined the length requirement of the 5′ single-stranded tail on the RNA template and the optimal reaction conditions for the unwinding activity of DpTV Hel. Moreover, DpTV Hel also contains NTPase activity. The ATPase activity of DpTV Hel could be significantly stimulated by dsRNA, and dsRNA could partially rescue the ATPase activity abolishment caused by mutations. Our study is the first to identify an alphatetravirus RNA helicase and further characterize its dsRNA unwinding and NTPase activities in detail and should foster our understanding of DpTV and other alphatetraviruses.

Expression, purification, and functional characterization of a stable helicase domain from a tomato mosaic virus replication protein

Tomato mosaic virus (genus, Tobamovirus) is a member of the alphavirus-like superfamily of positive-strand RNA viruses, which include many plant and animal viruses of agronomical and clinical importance. The RNA of alphavirus-like superfamily members encodes replication-associated proteins that contain a putative superfamily 1 helicase domain. To date, a viral three-dimensional superfamily 1 helicase structure has not been solved. For the study reported herein, we expressed tomato mosaic virus replication proteins that contain the putative helicase domain and additional upstream N-terminal residues in Escherichia coli. We found that an additional 155 residues upstream of the N-terminus of the helicase domain were necessary for stability. We developed an efficient procedure for the expression and purification of this fragment and have examined factors that affect its stability. Finally, we also showed that the stable fragment has nucleoside 5′-triphosphatase activity.

Inhibition of Hepatitis E virus replication using short hairpin RNA (shRNA)

Hepatitis E virus (HEV) is a non-enveloped, single-stranded, positive sense RNA virus, which is a major cause of water-borne hepatitis. RNA interference (RNAi) is a sequence-specific cellular antiviral defence mechanism, induced by double-stranded RNA, which we used to investigate knockdown of several genes and the 3′ cis-acting element (CAE) of HEV. In the present report, shRNAs were developed against the putative helicase and replicase domains and the 3′CAE region of HEV. Production of siRNA was confirmed by northern hybridization. The possible innate response induction due to shRNA expressions was verified by transcript analysis for interferon-β and 2′,5′-oligoadenylate synthetase genes and was found to be absent. Initially, the selected shRNAs were tested for their efficiency against the respective genes/3′CAE using inhibition of fused viral subgenomic target domain–renilla luciferase reporter constructs. The effective shRNAs were studied for their inhibitory effects on HEV replication in HepG2 cells using HEV replicon and reporter replicon. RNAi mediated silencing was demonstrated by reduction of luciferase activity in subgenomic target–reporter constructs and reporter replicon. The real time PCR was used to demonstrate inhibition of native replicon replication in transfected cells. Designed shRNAs were found to be effective in inhibiting virus replication to a variable extent (45–93%).

Clinical and mutational profile in spinal muscular atrophy with respiratory distress (SMARD): defining novel phenotypes through hierarchical cluster analysis

Autosomal recessive spinal muscular atrophy with respiratory distress (SMARD) is a heterogeneous disorder. Mutations in the immunoglobulin micro-binding protein gene (IGHMBP2) lead to SMARD1, but clinical criteria that delineate SMARD1 from other SMARD syndromes are not well established. Here we present a retrospective clinical and genetic study to determine the criteria that would predict the presence or absence of IGHMBP2 mutations. From 141 patients with respiratory distress and a spinal muscular atrophy phenotype we recorded the clinical features through a questionnaire and sequenced the entire coding region of IGHMBP2. In 47 (33%) patients we identified IGHMBP2 mutations, 14 of which were not described before. Clinical features and combinations thereof associated with the presence of IGHMBP2 mutations were discovered through hierarchical cluster analysis. This method detects common traits not evident at first sight by grouping items according to their similarity. The combination of "manifestation of respiratory failure between 6 weeks and 6 months" AND ("presence of diaphragmatic eventration" OR "preterm birth") predicted the presence of IGHMBP2 mutations with 98% sensitivity and 92% specificity. Non-SMARD1 patients fell into two different symptom clusters, mainly separated by the age at respiratory failure and the presence of multiple congenital contractures. The 14 novel IGHMBP2 mutations comprised missense, frameshift, splice-site, and nonsense mutations. All missense mutations altered conserved residues within or adjacent to the putative DNA helicase domain. The c.1235+3A>G splice-site mutation did not entirely suppress correct splicing and we found a residual wild-type IGHMBP2 mRNA steady-state level of 24.4+/-6.9%, which was, however, not sufficient to avert SMARD1 in this patient.

O.174 Subcellular localization of hepatitis C virus structural proteins in a cell culture system that efficiently replicates the virus

Two flavivirus-like viruses, GB virus-A (GBV-A) and GB virus-B (GBV-B), were recently identified in the GB hepatitis agent, and are distinct from the hepatitis A to E viruses. The putative helicase domain of GBV-A and GBV-B was found to have amino acid sequence homology with hepatitis C virus (HCV), and distantly, is also related to pestiviruses, flaviviruses, and plant viruses. A phylogenetic tree construction showed that GBVs and HCV are closely related, and they are clustered with pestiviruses, flaviviruses and plant viruses in that order.

A Novel Splicing Mutation in the Gene Encoding the Chromatin-Associated Factor ATRX Associated with Acquired Hemoglobin H Disease in Myelodysplastic Syndrome (ATMDS).

Acquired hemoglobin H (HbH) disease (α thalassemia) can arise in the setting of marrow neoplasia, especially the myelodysplastic syndrome (MDS). This association has been termed α thalassemia-MDS (ATMDS, MIM #300448). Recently, we have linked ATMDS to acquired point mutations in the ATRX gene, which encodes an X-linked chromatin-associated factor whose precise normal cellular function is unknown (Steensma et al, Blood 2004; 103:2019, and Gibbons et al, Nature Genetics 2003; 34:446).We now describe a novel acquired splicing mutation in the helicase domain of the ATRX gene in a 71 year-old man of southern Italian descent. The patient, who previously had normal blood indices, presented with a 6-month history of microcytic anemia (Hb 7.7 g/dL, MCV 73.7 fL, reticulocytes 0.2%; WBC 11,800/uL with 29% circulating blasts). Peripheral smear demonstrated marked anisopoikilocytosis and the presence of poorly hemoglobinized ghost cells, while a marrow aspirate and biopsy revealed dysplastic features including atypical megakaryocytes, and approximately 20% blasts, consistent with a diagnosis of MDS in leukemic transformation. On supravital staining, approximately 30% of circulating erythrocytes contained HbH inclusions. The α/β globin transcript ratio measured by quantitative real-time PCR ranged between 0.11 and 0.39, compared with the mean ratio for 11 normals (α/β ratios in normals ranged between 0.8–1.2 fold of the mean). Despite this evidence of α thalassemia, no gross rearrangements of the α globin cluster were detected on Southern blotting or by FISH, and sequencing of the α globin genes was unremarkable. Screening of the entire coding region of the ATRX gene by denaturing high-performance liquid chromatograpy showed no abnormalities. Amplification and sequencing of cDNA made from the patient's blood, however, revealed loss of ATRX exon 28 (in the middle of the putative helicase domain), resulting in a frame shift and generation of a premature stop codon. The causative mutation at the genomic level remains unknown, despite sequencing of the consensus splice sites framing both ATRX exon 28 and the surrounding regions. A clonally restricted mutation in a distant cryptic splice enhancer or inhibitor is suspected. Following treatment with idarubicin and Ara-C, the patient achieved a complete remission with normalization of red cell morphology and indices, and HbH was no longer detectable.This case emphasizes the need to search carefully for abnormal mRNA splicing when screening for ATRX mutations and provides further support for a link between ATRX mutations and ATMDS. Among 20 ATMDS patients analyzed molecularly, we have now characterized ATRX missense, null, and nonsense mutations in 9 patients, found a codon substitution of uncertain significance in 1 patient, and detected ATRX splicing mutations in 3 patients, including the present case. Additionally, one patient had an acquired α globin deletion limited to the neoplastic clone (Steensma et al, Blood 2004; 103:1518). The molecular defect remains unknown in 6 cases. Since RNA is not available from these 6 archival cases, it is possible that they have ATRX splicing mutations as well.

Allelic heterogeneity of SMARD1 at the IGHMBP2 locus.

Spinal Muscular Atrophy with Respiratory Distress (SMARD) is an autosomal recessive disorder characterized by neurogenic muscular atrophy due to progressive anterior horn cell degeneration and early life-threatening respiratory failure ascribed to diaphragmatic dysfunction. SMARD is clinically and genetically heterogeneous. SMARD type 1 is characterized by onset of respiratory failure within the first weeks of life and has been ascribed to mutations in the immunoglobulin mu-binding protein 2 (IGHMBP2) gene on chromosome 11q13-q21. We report here the identification of nine novel IGHMBP2 mutations in five SMARD1 patients, including seven missense [ c.587A>G (p.Gln196Arg), c.647C>T (p.Pro216Leu), c.752T>C (p.Leu251Pro), c.1693G>A (p.Asp565Asn), c.1730T>C (p.Leu577Pro), c.1807C>T (p.Arg603Cys), c.1909C>T (p.Arg637Cys)] and two nonsense mutations [ c.1488C>A (p.Cys496X), c.2368C>T (p.Arg790X)]. Interestingly, 7 of 9 mutations occurred at highly conserved residues of the putative DNA helicase domain. The identification of novel IGHMBP2 variants will hopefully help diagnosing SMARD1 and contribute to a better functional characterization of IGHMBP2 gene product.

Defining the structure-function relationships of bluetongue virus helicase protein VP6.

The VP6 protein of bluetongue virus possesses a number of activities, including nucleoside triphosphatase, RNA binding, and helicase activity (N. Stauber, J. Martinez-Costas, G. Sutton, K. Monastyrskaya, and P. Roy, J. Virol. 71:7220-7226, 1997). Although the enzymatic functions of the protein have been documented, a detailed structure and function study has not been completed and the oligomeric form of the protein in solution has not been described. In this study, we have characterized VP6 activity by creating site-directed mutations in the putative functional helicase domains. Mutant proteins were expressed at high levels in an insect cell by using recombinant baculoviruses purified and analyzed for ATP binding, ATP hydrolysis, and RNA unwinding activities. UV cross-linking experiments indicated that the lysine residue in the conserved motif AXXGXGK(110)V is directly involved in ATP binding, whereas mutant R(205)Q in the arginine-rich motif ER(205)XGRXXR bound ATP at a level comparable to that of the wild-type protein. The RNA binding activity was drastically altered in the R(205)Q mutant and was also affected in the K(110)N mutant. Helicase activity was altered in both mutants. The mutation E(157)N in the DEXX sequence, presumed to act as a Walker B motif, showed an intermediate activity, implying that this motif does not play a crucial role in VP6 function. Purified protein demonstrated stable oligomers with a ring-like morphology in the presence of nucleic acids similar to those shown by other helicases. Gel filtration chromatography, native gel electrophoresis, and glycerol gradient analysis clearly indicated multiple oligomeric forms of VP6.

Nucleotide sequence and genome organization of Cucurbit yellow stunting disorder virus RNA1.

The complete nucleotide sequence of Cucurbit yellow stunting disorder virus (CYSDV) RNA1, a member of the Crinivirus genus in the Closteroviridae, was determined. CYSDV RNA1 is 9126 nucleotides long and contains two overlapping open reading frames (ORFs) that encode the replication module, consisting of the putative papain-like cysteine proteinase, methyl transferase, helicase, and polymerase domains, a small 5 kDa hydrophobic protein and two further downstream ORFs potentially encoding proteins respectively 25 and 22 kDa in size. The genomic position and homology of the four domains comprising the replication module appear to be similar for all sequenced criniviruses but there is divergence in the downstream ORFs, in terms of number, size, position and sequence homology.

Structure-Function Analysis of Escherichia coli DNA Helicase I Reveals Non-overlapping Transesterase and Helicase Domains

TraI (DNA helicase I) is an Escherichia coli F plasmid-encoded protein required for bacterial conjugative DNA transfer. The protein is a sequence-specific DNA transesterase that provides the site- and strand-specific nick required to initiate DNA strand transfer and a 5' to 3' DNA helicase that unwinds the F plasmid to provide the single-stranded DNA that is transferred from donor to recipient. Sequence comparisons with other transesterases and helicases suggest that these activities reside in the N- and C-terminal regions of TraI, respectively. Computer-assisted secondary structure probability analysis identified a potential interdomain region spanning residues 304-309. Proteins encoded by segments of traI, whose N or C terminus either flanked or coincided with this region, were purified and assessed for catalytic activity. Amino acids 1-306 contain the transesterase activity, whereas amino acids 309-1504 contain the helicase activity. The C-terminal 252 amino acids of the 1756-amino acid TraI protein are not required for either helicase or transesterase activity. Protein and nucleic acid sequence similarity searches indicate that the occurrence of both transesterase- and helicase-associated motifs in a conjugative DNA transfer initiator protein is rare. Only two examples (other than R100 plasmid TraI) were found: R388 plasmid TrwC and R46 plasmid (pKM101) TraH, belonging to the IncW and IncN groups of broad host range conjugative plasmids, respectively. The most significant structural difference between these proteins and TraI is that TraI contains an additional region of approximately 650 residues between the transesterase domain and the helicase-associated motifs. This region is required for helicase activity.

Complete genome sequence and analyses of the subgenomic RNAs of sweet potato chlorotic stunt virus reveal several new features for the genus Crinivirus.

The complete nucleotide sequences of genomic RNA1 (9,407 nucleotides [nt]) and RNA2 (8,223 nt) of Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus, family Closteroviridae) were determined, revealing that SPCSV possesses the second largest identified positive-strand single-stranded RNA genome among plant viruses after Citrus tristeza virus. RNA1 contains two overlapping open reading frames (ORFs) that encode the replication module, consisting of the putative papain-like cysteine proteinase, methyltransferase, helicase, and polymerase domains. RNA2 contains the Closteroviridae hallmark gene array represented by a heat shock protein homologue (Hsp70h), a protein of 50 to 60 kDa depending on the virus, the major coat protein, and a divergent copy of the coat protein. This grouping resembles the genome organization of Lettuce infectious yellows virus (LIYV), the only other crinivirus for which the whole genomic sequence is available. However, in striking contrast to LIYV, the two genomic RNAs of SPCSV contained nearly identical 208-nt-long 3' terminal sequences, and the ORF for a putative small hydrophobic protein present in LIYV RNA2 was found at a novel position in SPCSV RNA1. Furthermore, unlike any other plant or animal virus, SPCSV carried an ORF for a putative RNase III-like protein (ORF2 on RNA1). Several subgenomic RNAs (sgRNAs) were detected in SPCSV-infected plants, indicating that the sgRNAs formed from RNA1 accumulated earlier in infection than those of RNA2. The 5' ends of seven sgRNAs were cloned and sequenced by an approach that provided compelling evidence that the sgRNAs are capped in infected plants, a novel finding for members of the Closteroviridae.

Functional crosstalk between hOgg1 and the helicase domain of Cockayne syndrome group B protein

We have previously reported that the Cockayne syndrome group B gene product (CSB) contributes to base excision repair (BER) of 8-hydroxyguanine (8-OH-Gua) and the importance of motifs V and VI of the putative helicase domains of CSB in BER of 8-OH-Gua. To further elucidate the function of CSB in BER, we investigated its role in the pathway involving human 8-OH-Gua glycosylase/apurinic lyase (hOgg1). Depletion of CSB protein with anti-CSB antibody reduced the 8-OH-Gua incision rate of wild type cell extracts but not of CSB null and motif VI mutant cell extracts, suggesting a direct contribution of CSB to the catalytic process of 8-OH-Gua incision and the importance of its motif VI in this pathway. Introduction of recombinant purified CSB partially complemented the depletion of CSB as shown by the recovery of the incision activity. This complementation could not fully recover the deficiency of the incision activity in WCE from CS-B null and mutant cell lines, suggesting that some additional factor(s) are necessary for the full activity. Electrophoretic mobility shift assays (EMSAs) showed a defect in binding of CSB null and motif VI mutant cell extracts to 8-OH-Gua-containing oligonucleotides. We detected less hOgg1 transcript and protein in the cell extracts from CS-B null and mutant cells, suggesting hOgg1 may be the missing component. Pull-down of hOgg1 by histidine-tagged CSB and co-localization of those two proteins after γ-radiation indicated their co-existence in vivo, particularly under cellular stress. However, we did not detect any functional and physical interaction between purified CSB and hOgg1 by incision, gel shift and yeast two-hybrid assays, suggesting that even though hOgg1 and CSB might be in a common protein complex, they may not interact directly. We conclude that CSB functions in the catalysis of 8-OH-Gua BER and in the maintenance of efficient hOgg1 expression, and that motif VI of the putative helicase domain of CSB is crucial in these functions.

The Cockayne Syndrome Group B Gene Product Is Involved in Cellular Repair of 8-Hydroxyadenine in DNA

Cockayne syndrome (CS) is a human disease characterized by sensitivity to sunlight, severe neurological abnormalities, and accelerated aging. CS has two complementation groups, CS-A and CS-B. The CSB gene encodes the CSB protein with 1493 amino acids. We previously reported that the CSB protein is involved in cellular repair of 8-hydroxyguanine, an abundant lesion in oxidatively damaged DNA and that the putative helicase motif V/VI of the CSB may play a role in this process. The present study investigated the role of the CSB protein in cellular repair of 8-hydroxyadenine (8-OH-Ade), another abundant lesion in oxidatively damaged DNA. Extracts of CS-B-null cells and mutant cells with site-directed mutation in the motif VI of the putative helicase domain incised 8-hydroxyadenine in vitro less efficiently than wild type cells. Furthermore, CS-B-null and motif VI mutant cells accumulated more 8-hydroxyadenine in their genomic DNA than wild type cells after exposure to gamma-radiation at doses of 2 or 5 Gy. These results suggest that the CSB protein contributes to cellular repair of 8-OH-Ade and that the motif VI of the putative helicase domain of CSB is required for this activity.