A Constitutively Expressed, Truncated umuDC Operon Regulates the recA-Dependent DNA Damage Induction of a Gene in Acinetobacter baylyi Strain ADP1

In response to environmentally caused DNA damage, SOS genes are up-regulated due to RecA-mediated relief of LexA repression. In Escherichia coli, the SOS umuDC operon is required for DNA damage checkpoint functions and for replicating damaged DNA in the error-prone process called SOS mutagenesis. In the model soil bacterium Acinetobacter baylyi strain ADP1, however, the content, regulation, and function of the umuDC operon are unusual. The umuC gene is incomplete, and a remnant of an ISEhe3-like transposase has replaced the middle 57% of the umuC coding region. The umuD open reading frame is intact, but it is 1.5 times the size of other umuD genes and has an extra 5′ region that lacks homology to known umuD genes. Analysis of a umuD::lacZ fusion showed that umuD was expressed at very high levels in both the absence and presence of mitomycin C and that this expression was not affected in a recA-deficient background. The umuD mutation did not affect the growth rate or survival after UV-induced DNA damage. However, the UmuD-like protein found in ADP1 (UmuDAb) was required for induction of an adjacent DNA damage-inducible gene, ddrR. The umuD mutation specifically reduced the DNA damage induction of the RecA-dependent DNA damage-inducible ddrR locus by 83% (from 12.9-fold to 2.3-fold induction), but it did not affect the 33.9-fold induction of benA, an unrelated benzoate degradation gene. These data suggest that the response of the ADP1 umuDC operon to DNA damage is unusual and that UmuDAb specifically regulates the expression of at least one DNA damage-inducible gene.     

The BRCT Domain of PARP-1 Is Required for Immunoglobulin Gene Conversion

Genetic variation at immunoglobulin (Ig) gene variable regions in B-cells is created through a multi-step process involving deamination of cytosine bases by activation-induced cytidine deaminase (AID) and their subsequent mutagenic repair. To protect the genome from dangerous, potentially oncogenic effects of off-target mutations, both AID activity and mutagenic repair are targeted specifically to the Ig genes. However, the mechanisms of targeting are unknown and recent data have highlighted the role of regulating mutagenic repair to limit the accumulation of somatic mutations resulting from the more widely distributed AID-induced lesions to the Ig genes. Here we investigated the role of the DNA damage sensor poly-(ADPribose)-polymerase-1 (PARP-1) in the repair of AID-induced DNA lesions. We show through sequencing of the diversifying Ig genes in PARP-1^−/− DT40 B-cells that PARP-1 deficiency results in a marked reduction in gene conversion events and enhanced high-fidelity repair of AID-induced lesions at both Ig heavy and light chains. To further characterize the role of PARP-1 in the mutagenic repair of AID-induced lesions, we performed functional analyses comparing the role of engineered PARP-1 variants in high-fidelity repair of DNA damage induced by methyl methane sulfonate (MMS) and the mutagenic repair of lesions at the Ig genes induced by AID. This revealed a requirement for the previously uncharacterized BRCT domain of PARP-1 to reconstitute both gene conversion and a normal rate of somatic mutation at Ig genes, while being dispensable for the high-fidelity base excision repair. From these data we conclude that the BRCT domain of PARP-1 is required to initiate a significant proportion of the mutagenic repair specific to diversifying antibody genes. This role is distinct from the known roles of PARP-1 in high-fidelity DNA repair, suggesting that the PARP-1 BRCT domain has a specialized role in assembling mutagenic DNA repair complexes involved in antibody diversification.     

cGMP Is Required for Gibberellic Acid-Induced Gene Expression in Barley Aleurone

The occurrence and roles of cGMP were investigated in aleurone layers and protoplasts isolated from barley (cv Himalaya) grain. Levels of cGMP in freshly isolated barley aleurone layers ranged from 0.065 to 0.08 pmol/g fresh weight of tissue, and cGMP levels increased transiently after incubation in gibberellic acid (GA). Abscisic acid (ABA) did not increase cGMP levels in aleurone layers. LY 83583 (LY), an inhibitor of guanylyl cyclase, prevented the GA-induced increase in cGMP and inhibited GA-induced [alpha]-amylase synthesis and secretion. The inhibitory effects of LY could be overcome by membrane-permeant analogs of cGMP. LY also prevented GA-induced accumulation of [alpha]-amylase and GAMYB mRNAs. cGMP alone was not sufficient to induce the accumulation of [alpha]-amylase or GAMYB mRNA. LY had a less dramatic effect on the accumulation of mRNAs encoding the ABA-responsive gene Rab21. We conclude that cGMP plays an important role in GA, but not ABA, signaling in the barley aleurone cell.     

The Pre-NH2-Terminal Domain of the Herpes Simplex Virus 1 DNA Polymerase Catalytic Subunit Is Required for Efficient Viral Replication

The catalytic subunit of herpes simplex virus 1 DNA polymerase (HSV-1 Pol) has been extensively studied; however, its full complement of functional domains has yet to be characterized. A crystal structure has revealed a previously uncharacterized pre-NH₂-terminal domain (residues 1 to 140) within HSV-1 Pol. Due to the conservation of the pre-NH₂-terminal domain within the herpesvirus Pol family and its location in the crystal structure, we hypothesized that this domain provides an important function during viral replication in the infected cell distinct from 5′-3′ polymerase activity. We identified three pre-NH₂-terminal Pol mutants that exhibited 5′-3′ polymerase activity indistinguishable from that of wild-type Pol in vitro: deletion mutants PolΔN43 and PolΔN52 that lack the extreme N-terminal 42 and 51 residues, respectively, and mutant PolA₆, in which a conserved motif at residues 44 to 49 was replaced with alanines. We constructed the corresponding pol mutant viruses and found that the polΔN43 mutant displayed replication kinetics similar to those of wild-type virus, while polΔN52 and polA₆ mutant virus infection resulted in an 8-fold defect in viral yield compared to that achieved with wild type and their respective rescued derivative viruses. Additionally, both polΔN52 and polA₆ viruses exhibited defects in viral DNA synthesis that correlated with the observed reduction in viral yield. These results strongly indicate that the conserved motif within the pre-NH₂-terminal domain is important for viral DNA synthesis and production of infectious virus and indicate a functional role for this domain.     

Methyl-CpG-Binding Domain Protein MBD7 Is Required for Active DNA Demethylation in Arabidopsis

Although researchers have established that DNA methylation and active demethylation are dynamically regulated in plant cells, the molecular mechanism for the regulation of active DNA demethylation is not well understood. By using an Arabidopsis (Arabidopsis thaliana) line expressing the Promoter RESPONSIVE TO DEHYDRATION 29A:LUCIFERASE (ProRD29A:LUC) and Promoter cauliflower mosaic virus 35S:NEOMYCIN PHOSPHOTRANSFERASE II (Pro35S:NPTII) transgenes, we isolated an mbd7 (for methyl-CpG-binding domain protein7) mutant. The mbd7 mutation causes an inactivation of the Pro35S:NPTII transgene but does not affect the expression of the ProRD29A:LUC transgene. The silencing of the Pro35S:NPTII reporter gene is associated with DNA hypermethylation of the reporter gene. MBD7 interacts physically with REPRESSOR OF SILENCING5/INCREASED DNA METHYLATION2, a protein in the small heat shock protein family. MBD7 prefers to target the genomic loci with high densities of DNA methylation around chromocenters. The Gypsy-type long terminal repeat retrotransposons mainly distributed around chromocenters are most affected by mbd7 in all transposons. Our results suggest that MBD7 is required for active DNA demethylation and antisilencing of the genomic loci with high densities of DNA methylation in Arabidopsis.DNA methylation is an important epigenetic marker for genome stability and the regulation of gene expression in both plants and animals (Law and Jacobsen, 2010; He et al., 2011). In plants, the molecular mechanisms for DNA methylation have been well characterized by the use of powerful genetic screening systems (Bartee et al., 2001; Lindroth et al., 2001; Matzke et al., 2004; He et al., 2009). A transgene or an endogenous gene may be silenced because of DNA hypermethylation in the promoter region. Screenings for mutants with release of the silenced marker genes have identified many components that are involved in RNA-directed DNA methylation (RdDM) and in maintaining DNA methylation (Matzke and Birchler, 2005; Law and Jacobsen, 2009; He et al., 2011; Bender, 2012). DNA methylation is catalyzed by DNA methyltransferases including DNA METHYLTRANSFERASE1 (MET1) and CHROMOMETHYLASE3 (CMT3), which maintain symmetric CG and CHG methylation, respectively, during DNA replication, and DOMAINS REARRANGED METHYLASE2 (DRM2) and CMT2, which are required for establishing CHG and asymmetric CHH methylation during each cell cycle. DRM2 also catalyzes CG methylation (Law and Jacobsen, 2010; Haag and Pikaard, 2011; He et al., 2011; Zemach et al., 2013; Stroud et al., 2014). Twenty-four-nucleotide small RNAs produced through the RdDM pathway target genomic regions to guide the establishment of DNA methylation by DRM2 (Cao et al., 2003).DNA methylation can be actively removed by a subfamily of bifunctional DNA glycosylases/lyases including REPRESSOR OF SILENCING1 (ROS1; Gong et al., 2002) and its paralogs DEMETER and DEMETER-LIKE2/3 (Gehring et al., 2006; Ortega-Galisteo et al., 2008). DNA methylation can also be passively lost during DNA replication when DNA methylation cannot be maintained (Zhu, 2009). Promoter RESPONSIVE TO DEHYDRATION 29A:LUCIFERASE (ProRD29A:LUC) in the ProRD29A:LUC/Promoter cauliflower mosaic virus 35S:NEOMYCIN PHOSPHOTRANSFERASE II (Pro35S:NPTII) transgenic Arabidopsis (Arabidopsis thaliana) line has been used as a marker to identify ros1 and ros3 mutants in which both ProRD29A:LUC and Pro35S:NPTII are silenced (Gong et al., 2002; Zheng et al., 2008). ROS3 is an RNA-binding protein that facilitates the function of ROS1 in active DNA demethylation at certain genomic loci. Using Pro35S:NPTII as a selection marker for kanamycin-sensitive mutants and the 35S-SUC2 transgene or a chop PCR marker for assaying DNA methylation at the 3′ region of At1g26400 from transfer DNA (T-DNA) insertion mutants, researchers recently identified two genes involved in active DNA demethylation: ROS4/INCREASED DNA METHYLATION1 (IDM1) and ROS5/IDM2 (Li et al., 2012; Qian et al., 2012, 2014; Zhao et al., 2014). ROS4/IDM1 is a plant homeodomain-finger domain-containing histone acetyltransferase that catalyzes histone H3 lysine18 (H3K18) and lysine23 (H3K23) acetylation (Li et al., 2012; Qian et al., 2012). ROS5/IDM2 is a member of the small heat shock protein family that interacts physically with ROS4/IDM1 for the regulation of active DNA demethylation. Genetic analysis indicates that ROS1, ROS4/IDM1, and ROS5/IDM2 are in the same genetic pathway and that ROS4/IDM1 and ROS5/IDM2 may form a protein complex for the regulation of active DNA demethylation (Qian et al., 2014; Zhao et al., 2014).During the genetic screening for kanamycin-sensitive mutants using the ProRD29A:LUC/Pro35S:NPTII transgenic line in this study, we identified another mutant, mbd7, where the Pro35S:NPTII transgene is specifically silenced. MBD7 is a methyl-CpG-binding domain (MBD) protein containing three MBD motifs that bind in vitro to methylated symmetric CG sites. MBD7 localizes to all highly CpG-methylated chromocenters in vivo (Zemach and Grafi, 2003; Zemach et al., 2008). Recruitment of MBD7 to chromocenters is disrupted in decrease in DNA methylation1 (ddm1) and met1, two mutants with great reductions in DNA methylation, suggesting that DNA methylation is required for proper MBD7 localization (Zemach et al., 2005). In this study, we found that MBD7 interacts physically with ROS5/IDM2 and is required for the active DNA demethylation of certain genomic loci, especially for the Gypsy-type long terminal repeat (LTR) retrotransposons with high densities of DNA methylation around chromocenters in Arabidopsis.     

Residues in the N-Terminal Domain of MutL Required for Mismatch Repair in Bacillus subtilis

Mismatch repair is a highly conserved pathway responsible for correcting DNA polymerase errors incorporated during genome replication. MutL is a mismatch repair protein known to coordinate several steps in repair that ultimately results in strand removal following mismatch identification by MutS. MutL homologs from bacteria to humans contain well-conserved N-terminal and C-terminal domains. To understand the contribution of the MutL N-terminal domain to mismatch repair, we analyzed 14 different missense mutations in Bacillus subtilis MutL that were conserved with missense mutations identified in the human MutL homolog MLH1 from patients with hereditary nonpolyposis colorectal cancer (HNPCC). We characterized missense mutations in or near motifs important for ATP binding, ATPase activity, and DNA binding. We found that 13 of the 14 missense mutations conferred a substantial defect to mismatch repair in vivo, while three mutant alleles showed a dominant negative increase in mutation frequency to wild-type mutL. We performed immunoblot analysis to determine the relative stability of each mutant protein in vivo and found that, although most accumulated, several mutant proteins failed to maintain wild-type levels, suggesting defects in protein stability. The remaining missense mutations located in areas of the protein important for DNA binding, ATP binding, and ATPase activities of MutL compromised repair in vivo. Our results define functional residues in the N-terminal domain of B. subtilis MutL that are critical for mismatch repair in vivo.     

Myc Is Required for Activation of the ATM-Dependent Checkpoints in Response to DNA Damage

Background

The MYC protein controls cellular functions such as differentiation, proliferation, and apoptosis. In response to genotoxic agents, cells overexpressing MYC undergo apoptosis. However, the MYC-regulated effectors acting upstream of the mitochondrial apoptotic pathway are still unknown.

Principal Findings

In this study, we demonstrate that expression of Myc is required to activate the Ataxia telangiectasia mutated (ATM)-dependent DNA damage checkpoint responses in rat cell lines exposed to ionizing radiation (IR) or the bacterial cytolethal distending toxin (CDT). Phosphorylation of the ATM kinase and its downstream effectors, such as histone H2AX, were impaired in the myc null cell line HO15.19, compared to the myc positive TGR-1 and HOmyc3 cells. Nuclear foci formation of the Nijmegen Breakage Syndrome (Nbs) 1 protein, essential for efficient ATM activation, was also reduced in absence of myc. Knock down of the endogenous levels of MYC by siRNA in the human cell line HCT116 resulted in decreased ATM and CHK2 phosphorylation in response to irradiation. Conversely, cell death induced by UV irradiation, known to activate the ATR-dependent checkpoint, was similar in all the cell lines, independently of the myc status.

Conclusion

These data demonstrate that MYC contributes to the activation of the ATM-dependent checkpoint responses, leading to cell death in response to specific genotoxic stimuli.     

The Ligand Binding Domain of GCNF Is Not Required for Repression of Pluripotency Genes in Mouse Fetal Ovarian Germ Cells

In mice, successful development and reproduction require that all cells, including germ cells, transition from a pluripotent to a differentiated state. This transition is associated with silencing of the pluripotency genes Oct4 and Nanog. Interestingly, these genes are repressed at different developmental timepoints in germ and somatic cells. Ovarian germ cells maintain their expression until about embryonic day (E) 14.5, whereas somatic cells silence them much earlier, at about E8.0. In both somatic cells and embryonic stem cells, silencing of Oct4 and Nanog requires the nuclear receptor GCNF. However, expression of the Gcnf gene has not been investigated in fetal ovarian germ cells, and whether it is required for silencing Oct4 and Nanog in that context is not known. Here we demonstrate that Gcnf is expressed in fetal ovarian germ cells, peaking at E14.5, when Oct4 and Nanog are silenced. However, conditional ablation of the ligand-binding domain of Gcnf using a ubiquitous, tamoxifen-inducible Cre indicates that Gcnf is not required for the down-regulation of pluripotency genes in fetal ovarian germ cells, nor is it required for initiation of meiosis and oogenesis. These results suggest that the silencing of Oct4 and Nanog in germ cells occurs via a different mechanism from that operating in somatic cells during gastrulation.     

Gene A Protein of Bacteriophage S13 Is Required for Single-Stranded DNA Synthesis

The product of gene A of the small icosahedral DNA phage S13 has been found to be needed for single-stranded DNA synthesis in vivo in addition to its previously known role in progeny replicative-form DNA synthesis.     

A Conserved Interaction between the SDI Domain of Bre2 and the Dpy-30 Domain of Sdc1 Is Required for Histone Methylation and Gene Expression

In Saccharomyces cerevisiae, lysine 4 on histone H3 (H3K4) is methylated by the Set1 complex (Set1C or COMPASS). Besides the catalytic Set1 subunit, several proteins that form the Set1C (Swd1, Swd2, Swd3, Spp1, Bre2, and Sdc1) are also needed to mediate proper H3K4 methylation. Until this study, it has been unclear how individual Set1C members interact and how this interaction may impact histone methylation and gene expression. In this study, Bre2 and Sdc1 are shown to directly interact, and it is shown that the association of this heteromeric complex is needed for proper H3K4 methylation and gene expression to occur. Interestingly, mutational and biochemical analysis identified the C terminus of Bre2 as a critical protein-protein interaction domain that binds to the Dpy-30 domain of Sdc1. Using the human homologs of Bre2 and Sdc1, ASH2L and DPY-30, respectively, we demonstrate that the C terminus of ASH2L also interacts with the Dpy-30 domain of DPY-30, suggesting that this protein-protein interaction is maintained from yeast to humans. Because of the functionally conserved nature of the C terminus of Bre2 and ASH2L, this region was named the SDI (Sdc1 Dpy-30 interaction) domain. Finally, we show that the SDI-Dpy-30 domain interaction is physiologically important for the function of Set1 in vivo, because specific disruption of this interaction prevents Bre2 and Sdc1 association with Set1, resulting in H3K4 methylation defects and decreases in gene expression. Overall, these and other mechanistic studies on how H3K4 methyltransferase complexes function will likely provide insights into how human MLL and SET1-like complexes or overexpression of ASH2L leads to oncogenesis.     

耐热DNA聚合酶基因的克隆及在大肠杆菌中的表达

用PCR法从水生栖热菌菌株YT－1中扩增耐热DNA聚合酶基因,得到2．5kb的DNA片段t扩增片段重组到pUCl8中测序证实为Taq DNA聚合酶基因,将该片段重组到pBV221温控表达质粒中,在大肠杆菌中表达出94kDa的重组蛋白,100ml培养物的细胞产酶为1．5×10⁵u,表达的蛋白能催化PCR反应的进行。     

Taq DNA耐热聚合酶在大肠杆菌中的克隆和高表达

从水生栖热菌(Thermus aquaticus)YT-1中分离得到的Taq DNA 聚合酶是一种广泛应用于PCR的耐热DNA聚合酶。由于天然菌株酶产量较低,培养条件要求严格,酶的纯化过程极为繁琐而使产品成本较高,因而促使人们构建适合于大规模生产的基因工程菌株。已有人分别通过不同的途径,使用不同的载体,成功地在大肠杆菌菌株中表达了Taq 耐热DNA聚合酶基因。我们通过与前人不同的途径,把这一基因克隆到大肠杆菌的载体质粒pJLA503上并使其得以高表达,为降低生产成本和进一步研究该酶的各种特性提供了有利条件。     

The Carboxy-Terminal Segment of the Human Cytomegalovirus DNA Polymerase Accessory Subunit UL44 Is Crucial for Viral Replication

The amino-terminal 290 residues of UL44, the presumed processivity factor of human cytomegalovirus DNA polymerase, possess all of the established biochemical activities of the full-length protein, while the carboxy-terminal 143 residues contain a nuclear localization signal (NLS). We found that although the amino-terminal domain was sufficient for origin-dependent synthesis in a transient-transfection assay, the carboxy-terminal segment was crucial for virus replication and for the formation of DNA replication compartments in infected cells, even when this segment was replaced with a simian virus 40 NLS that ensured nuclear localization. Our results suggest a role for this segment in viral DNA synthesis.Human cytomegalovirus (HCMV) encodes a DNA polymerase which is composed of two subunits, UL54, the catalytic subunit, and UL44, an accessory protein (8, 12, 21). UL44 can be divided into two regions, a 290-residue amino (N)-terminal domain and a 143-residue carboxy (C)-terminal segment. The overall fold of the N-terminal domain is markedly similar to that of processivity factors such as herpes simplex virus type 1 (HSV-1) UL42 and eukaryotic proliferating cell nuclear antigen (6, 22, 41), which function to tether catalytic subunits to DNA to ensure long-chain DNA synthesis. In vitro, the N-terminal domain of UL44 is sufficient for all of the established biochemical activities of full-length UL44, including dimerization, binding to double-stranded DNA, interaction with UL54, and stimulation of long-chain DNA synthesis, consistent with a role as a processivity factor (4, 5, 8, 11, 23, 24, 39). In contrast, little is known about the functions of the C-terminal segment of UL44 other than its having been reported from transfection experiments to be important for downregulation of transactivation of a non-HCMV promoter (7) and to contain a nuclear localization signal (NLS) (3). Neither the importance of this NLS nor the role of the entire C-terminal segment has been investigated in HCMV-infected cells.We first examined whether the N-terminal domain is sufficient to support DNA synthesis from HCMV oriLyt in cells using a previously described cotransfection-replication assay (27, 28). A DpnI-resistant fragment, indicative of oriLyt-dependent DNA synthesis, was detected in the presence of wild-type (WT) UL44 (pSI-UL44) (34) and in the presence of the UL44 N-terminal domain (pSI-UL44ΔC290), but not in the presence of UL44-F121A (6, 34), a mutant form previously shown not to support oriLyt-dependent DNA synthesis (34) (Fig. ​(Fig.1A).1A). Thus, the N-terminal domain alone is sufficient to support oriLyt-dependent DNA synthesis in a transient-transfection assay.Open in a separate windowFIG. 1.Effects of UL44 C-terminal truncations in various assays. (A) HFF cells were cotransfected with the pSP50 plasmid (containing the oriLyt DNA replication origin), a plasmid expressing WT or mutant UL44 (as indicated at the top of the panel), and plasmids expressing all of the other essential HCMV DNA replication proteins. At 5 days posttransfection, total DNA was extracted and cleaved with DpnI to digest unreplicated DNA and a Southern blot assay was performed to detect replicated pSP50. An arrow indicates DpnI-resistant, newly synthesized pSP50 fragments. (B) FLAG-tagged constructs analyzed in panel C are cartooned as horizontal bars. The names of the constructs are above the bars. The lengths of the constructs in amino acids are indicated by the scale at the bottom of the panel. The positions of residues required but not necessarily sufficient for features of the constructs are designated by shading, as indicated at the bottom of the panel. (C) Vero cells were transfected with plasmids expressing WT UL44 (parts a to c), FLAG-UL44 (parts d to f), FLAG-UL44-290stop (parts g to i), or FLAG-UL44-290NLSstop (parts j to l). At 48 h posttransfection, cells were fixed and stained with 4′,6-diamidino-2-phenylindole (DAPI) to visualize the nucleus (blue) (parts a, d, g, and j) and by IF with anti-UL44 (part b) or anti-FLAG (parts e, h, and k) and a secondary antibody conjugated with Alexa 488 (green). Parts c, f, i, and l are merged from images in the left and middle columns. Magnification: ×1,000. (D) Replication kinetics of rescued viruses. Rescued derivatives of UL44 mutant viruses (UL44-290stop-R and UL44-290NLSstop-R) or WT AD169 viruses were used to infect HFF cells at an MOI of 1 PFU/cell. The supernatants from infected cells were collected every 24 h, and viral titers were determined by plaque assays on HFF cells.These results were somewhat unexpected, as the C-terminal segment contains a functional NLS identified in transfection assays (3). We therefore assayed the intracellular localization of WT and mutant UL44 following transient transfection using pcDNA3-derived expression plasmids. Since the anti-UL44 antibodies that we have tested do not recognize the N-terminal domain of UL44, we constructed UL44 genes to encode N-terminally FLAG-tagged full-length UL44 (FLAG-UL44) or a FLAG-tagged N-terminal domain, the latter by inserting three in-frame tandem stop codons after codon 290 (FLAG-UL44-290stop, Fig. ​Fig.1B).1B). We also constructed a mutant form encoding a FLAG-tagged N-terminal domain, followed by the simian virus 40 (SV40) T-antigen NLS (15-17), followed by three tandem stop codons (FLAG-UL44-290NLSstop, Fig. ​Fig.1B).1B). Vero cells were transfected with each construct using Lipofectamine 2000, fixed with 4% formaldehyde at 48 h posttransfection, and assayed by indirect immunofluorescence (IF) using anti-UL44 (Virusys) or anti-FLAG antibody (Sigma). We observed mostly nuclear localization of WT UL44 or FLAG-UL44 with either diffuse or more localized intranuclear distribution (Fig. ​(Fig.1C,1C, parts a to c and d to f, respectively) and some occasional perinuclear staining, which may be due to protein overexpression. In cells expressing FLAG-UL44-290NLSstop, we observed mostly diffuse nuclear localization with little to no perinuclear staining (Fig. ​(Fig.1C,1C, parts j to l). In cells expressing FLAG-UL44-290stop, we observed mostly cytoplasmic staining, but with some cells exhibiting some nuclear staining (Fig. ​(Fig.1C,1C, parts g to i), which may explain the ability of truncated UL44 to support oriLyt-dependent DNA replication in a transient-transfection assay (Fig. ​(Fig.1A1A).We next investigated whether the C-terminal segment of UL44 is necessary for viral replication. We reasoned that we could investigate whether any requirement for this segment could be due to a requirement for an NLS by testing whether the SV40 NLS could substitute for the loss of the UL44 C terminus. We therefore constructed HCMV UL44 mutant viruses by introducing the UL44-290stop and UL44-290NLSstop mutations into a WT AD169 bacterial artificial chromosome (BAC) using two-step red-mediated recombination as previously described (35, 38). We also constructed the same mutants with a FLAG epitope at the N terminus of UL44 (BAC-FLAG-UL44-290stop and BAC-FLAG-UL44-290NLSstop) to monitor UL44 expression, and we constructed rescued derivatives of the mutant BACs by replacing the mutated sequences with WT UL44 sequences, as described previously (35). We introduced BACs into human foreskin fibroblast (HFF) cells using electroporation (35, 38). In several experiments using at least two independent clones for each mutant, cells electroporated with any of the mutant BACs did not exhibit any cytopathic effect (CPE) within 21 days. In contrast, within 7 to 10 days, cells electroporated with the WT AD169 BAC, a BAC expressing WT UL44 with an N-terminal FLAG tag [AD169-BACF44 (35)], or any of the rescued derivatives began displaying a CPE and yielded infectious virus. The rescued derivatives of the nontagged mutants displayed replication kinetics similar to those of the WT virus following infection at a multiplicity of infection (MOI) of 1 PFU/cell (Fig. ​(Fig.1D).1D). The rescued derivatives of the FLAG-tagged mutants also replicated to WT levels (data not shown). Thus, the replication defects of the mutants were due to the introduced mutations that result in truncated UL44 either with or without the SV40 NLS. We therefore conclude that the C-terminal segment of UL44 is required for viral replication.To investigate the stage of viral replication at which the UL44 C-terminal segment is important, we first assayed the subcellular localization of immediate-early proteins IE1 and IE2 and FLAG-UL44 in cells electroporated with BAC DNA expressing the FLAG-tagged WT or the two mutant UL44s using IF at 2 days postelectroporation. IE1/IE2 could be detected diffusely distributed in nuclei of cells electroporated with all three BACs (Fig. 2b, f, and j). In cells electroporated with AD169-BACF44 or BAC-FLAG-UL44-290NLSstop, FLAG-UL44 was localized largely within the nucleus (Fig. 2c and k, respectively). In contrast, in cells electroporated with BAC-FLAG-UL44-290stop, the FLAG epitope was mainly localized diffusely in the cytoplasm, with only a small amount diffusely distributed in the nucleus (Fig. ​(Fig.2g).2g). These data indicate that IE proteins expressed from mutant BACs are properly localized and suggest that without its C-terminal segment, which includes the NLS identified in transfection assays (3), UL44 cannot efficiently localize to the nucleus in HCMV-infected cells. However, addition of the SV40 NLS was sufficient to efficiently localize the N-terminal domain of UL44 to the nucleus. Thus, the requirement for the C-terminal segment of UL44 for viral replication is not due solely to its NLS.Open in a separate windowFIG. 2.Localization of IE1/IE2 and FLAG-UL44 proteins in electroporated cells. HFF cells were electroporated with AD169-BACF44 (panels a to d), BAC-UL44-290stop (panels e to h), or BAC-FLAG-UL44-290NLSstop (panels i to l). At 48 h posttransfection, cells were fixed and probed with anti-IE1/2 (Virusys) or anti-FLAG (Sigma). Secondary antibodies coupled to fluorophores were used for visualization of IE1/2 (anti-mouse Alexa 594; panels b, f, and j) and FLAG (anti-rabbit Alexa 488; panels c, g, and k) antibodies. DAPI was used to counterstain the nucleus (panels a, e, and i). Panels d, h, and l are merged images of the panels in the other columns. Magnification: ×1,000.We next investigated if the block in viral replication due to the loss of the C-terminal segment could be attributed to a defect in viral DNA synthesis. Cells were electroporated with AD169-BACF44 or BAC-FLAG-UL44-290NLSstop, and viral DNA accumulation was assayed by quantitative real-time PCR at various times postelectroporation (Fig. ​(Fig.3)3) as previously described (32, 35). In HFFs electroporated with AD169-BACF44, viral DNA began to accumulate above the input levels by 8 days postelectroporation and increased over time, with as much as a 350-fold increase over the input DNA level by 18 days postelectroporation. In contrast, levels of viral DNA in cells electroporated with BAC-UL44-290NLSstop did not increase above input levels, even by 18 days postelectroporation. These data are consistent with the notion that the UL44 C-terminal segment is required for viral DNA synthesis, although we caution that the assay did not detect DNA synthesis from AD169-BACF44 until day 8, when viral spread had likely occurred (see below).Open in a separate windowFIG. 3.Quantification of viral DNA accumulation in electroporated cells. HFF cells were electroporated with AD169-BACF44 or BAC-FLAG-UL44-290NLSstop, and total DNA was harvested on the days postelectroporation indicated. Viral DNA accumulation was assessed by real-time PCR by assessing levels of the UL83 gene and normalizing to levels of the cellular β-actin gene (32). The data are presented as the fold increase in normalized viral DNA levels over the amount of input DNA (day 1).We also analyzed the localization patterns of UL44 and UL57, the viral single-stranded DNA binding protein, which is a marker for viral DNA replication compartments (1, 2, 18, 26, 29). At 8 days postelectroporation with AD169-BACF44, UL57 and FLAG-UL44 largely colocalized within a single large intranuclear structure that likely represents a fully formed replication compartment, with some cells containing multiple smaller globular structures within the nucleus that likely represent earlier stages of replication compartments (1, 2, 29) (Fig. 4a to d). Neighboring cells also stained for UL57 and FLAG-UL44, indicative of viral spread. In contrast, in cells electroporated with BAC-FLAG-UL44-290NLSstop, UL57 (Fig. ​(Fig.4f)4f) was found in either punctate or small globular structures. This pattern of UL57 staining resembled that observed at very early stages of viral DNA synthesis in HCMV-infected cells, but the structures were larger and less numerous than those observed in HCMV-infected cells in the presence of a viral DNA polymerase inhibitor (2, 29). Staining for FLAG-UL44 was nuclear and largely diffuse, with some areas of more concentrated staining (Fig. ​(Fig.4g),4g), which could also be observed in some cells at day 2 postelectroporation (Fig. ​(Fig.3k).3k). This pattern of UL44 localization was generally similar to that observed in HCMV-infected cells at very early stages of infection or when HCMV DNA synthesis is blocked and also similar to the pattern in cells transfected with a UL84 null mutant BAC (2, 29, 33, 40). Importantly, little colocalization of UL57 and UL44 was observed, with areas of concentration of UL57 or UL44 occupying separate regions in the nuclei of these cells (Fig. ​(Fig.4h).4h). We are unaware of any other examples of this pattern of localization of these proteins in HCMV-infected cells and suggest that it may be a result of the loss of the UL44 C-terminal segment. These results indicate that this segment is important for efficient formation of viral DNA replication compartments, again consistent with a requirement for this portion of UL44 for viral DNA synthesis.Open in a separate windowFIG. 4.Localization of UL57 and FLAG-UL44 proteins in electroporated cells. HFF cells were electroporated with AD169-BACF44 (panels a to d) or BAC-FLAG-UL44-290NLSstop (panels e to h). At 8 days posttransfection, cells were fixed and then stained with antibodies specific for UL57 (Virusys) or FLAG (Sigma), followed by a secondary antibody coupled to fluorophores to detect UL57 (anti-mouse Alexa 594; panels b and f) and FLAG (anti-rabbit Alexa 488; panels c and g) antibodies. DAPI stain was used to counterstain the nucleus (panels a and e). Panels d and h are merged images of the panels in the other columns. White arrows identify punctate UL57 staining. Yellow arrows identify areas of concentration of FLAG-UL44 staining. Magnification: ×1,000.Our results, taken together, argue for a role for the C-terminal segment of UL44 in HCMV-infected cells in efficient nuclear localization of UL44 and a role in viral DNA synthesis beyond its role in nuclear localization. It is possible that this segment interacts with host or viral proteins involved in DNA replication. Of the various proteins reported to interact with UL44 (10, 19, 30, 31, 35-37), interesting candidates include the host protein nucleolin, which has been shown to associate with UL44 and be important for viral DNA synthesis (35), and the viral UL112-113 proteins, which in transfection assays were shown to recruit UL44 to early sites of DNA replication (2, 29, 33). After this paper was submitted, Kim and Ahn reported that the C-terminal segment of UL44 is necessary for interaction with a UL112-113 protein and, similar to our findings, crucial for viral replication (19). However, contrary to our findings, they reported that this segment was not necessary for efficient nuclear localization of UL44 (19). It may well be that the C-terminal segment of UL44 also has some other role later in viral replication, perhaps in gene expression, as has been suggested (7, 13, 14).A virus with a deletion of the C-terminal 150 amino acids of the HSV-1 polymerase accessory subunit UL42 displays no obvious defect in replication (9). Thus, it appears that HSV-1 and HCMV exhibit different requirements for the C-terminal segments of their respective accessory proteins. This and many other differences between these functionally and structurally orthologous proteins (5, 6, 20, 24, 25) suggest considerable selection for different features during evolution.