共查询到20条相似文献,搜索用时 15 毫秒
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Michael West David Flanery Kelly Woytek Dhandapani Rangasamy Van G. Wilson 《Journal of virology》2001,75(24):11948-11960
Bovine papillomavirus type 1 (BPV-1) requires viral proteins E1 and E2 for efficient DNA replication in host cells. E1 functions at the BPV origin as an ATP-dependent helicase during replication initiation. Previously, we used alanine mutagenesis to identify two hydrophilic regions of the E1 DNA binding domain (E1DBD), HR1 (E1(179-191)) and HR3 (E1(241-252)), which are critical for sequence-specific recognition of the papillomavirus origin. Based on sequence and structure, these regions are similar in spacing and location to DNA binding regions A and B2 of T antigen, the DNA replication initiator of simian virus 40 (SV40). HR1 and A are both part of extended loops which are supported by residues from the HR3 and B2 alpha-helices. Both elements contain basic residues which may contact DNA, although lack of cocrystal structures for both E1 and T antigen make this uncertain. To better understand how E1 interacts with origin DNA, we used random mutagenesis and a yeast one-hybrid screen to select mutations of the E1DBD which disrupt sequence-specific DNA interactions. From the screen we selected seven single point mutants and one double point mutant (F175S, N184Y/K288R, D185G, V193M, F237L, K241E, R243K, and V246D) for in vitro analysis. All mutants tested in electrophoretic mobility shift assays displayed reduced sequence-specific DNA binding compared to the wild-type E1DBD. Mutants D185G, F237L, and R243K were rescued in vitro for DNA binding by the replication enhancer protein E2. We also tested the eight mutations in full-length E1 for the ability to support DNA replication in Chinese hamster ovary cells. Only mutants D185G, F237L, and R243K supported significant DNA replication in vivo which highlights the importance of E1DBD-E2 interactions for papillomavirus DNA replication. Based on the specific point mutations examined, we also assigned putative roles to individual residues in DNA binding. Finally, we discuss sequence and spacing similarities between E1 HR1 and HR3 and short regions of two other DNA tumor virus origin-binding proteins, SV40 T antigen and Epstein-Barr virus nuclear antigen 1 (EBNA1). We propose that all three proteins use a similar DNA recognition mechanism consisting of a loop structure which makes base-specific contacts (HR1) and a helix which primarily contacts the DNA backbone (HR3). 相似文献
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Differential Requirements for Conserved E2 Binding Sites in the Life Cycle of Oncogenic Human Papillomavirus Type 31 总被引:4,自引:2,他引:2
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Human papillomavirus (HPV) E2 proteins regulate viral replication by binding to sites in the upstream regulatory region (URR) and by complex formation with the E1 origin recognition protein. In the genital HPV types, the distribution and location of four E2 binding sites (BS1 to BS4) which flank a single E1 binding site are highly conserved. We have examined the roles of these four E2 sites in the viral life cycle of HPV type 31 (HPV31) by using recently developed methods for the biosynthesis of papillomaviruses from transfected DNA templates (M. G. Frattini et al., Proc. Natl. Acad. Sci. USA 93:3062–3067, 1996). In transient assays, no single site was found to be necessary for replication, and mutation of the early promoter-proximal site (BS4) led to a fourfold increase in replication. Cotransfection of the HPV31 wild-type (HPV-wt) and mutant genomes with expression vectors revealed that E1 stimulated replication of HPV31-wt as well as the HPV31-BS1, -BS2, and -BS3 mutants. In contrast, increased expression of E2 decreased replication of these genomes. Replication of the HPV31-BS4 mutant genome was not further increased by cotransfection of E1 expression vectors but was stimulated by E2 coexpression. In stably transfected normal human keratinocytes, mutation of either BS1, BS3, or BS4 resulted in integration of viral genomes into host chromosomes. In contrast, mutation of BS2 had no effect on stable maintenance of episomes or copy number. Following growth of stably transfected lines in organotypic raft cultures, the differentiation-dependent induction of late gene expression and amplification of viral DNA of the BS2 mutant was found to be similar to that of HPV31-wt. We were unable to find a role for BS2 in our assays for viral functions. We conclude that at least three of the four E2 binding sites in the URRs of HPVs are essential for the productive viral life cycle. The specific arrangement of E2 binding sites within the URR appears to be more important for viral replication than merely the number of sites. 相似文献
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Mart Reimund Mikael Larsson Oleg Kovrov Sergo Kasvandik Gunilla Olivecrona Aivar Lookene 《The Journal of biological chemistry》2015,290(22):13919-13934
GPIHBP1 is an endothelial membrane protein that transports lipoprotein lipase (LPL) from the subendothelial space to the luminal side of the capillary endothelium. Here, we provide evidence that two regions of GPIHBP1, the acidic N-terminal domain and the central Ly6 domain, interact with LPL as two distinct binding sites. This conclusion is based on comparative binding studies performed with a peptide corresponding to the N-terminal domain of GPIHBP1, the Ly6 domain of GPIHBP1, wild type GPIHBP1, and the Ly6 domain mutant GPIHBP1 Q114P. Although LPL and the N-terminal domain formed a tight but short lived complex, characterized by fast on- and off-rates, the complex between LPL and the Ly6 domain formed more slowly and persisted for a longer time. Unlike the interaction of LPL with the Ly6 domain, the interaction of LPL with the N-terminal domain was significantly weakened by salt. The Q114P mutant bound LPL similarly to the N-terminal domain of GPIHBP1. Heparin dissociated LPL from the N-terminal domain, and partially from wild type GPIHBP1, but was unable to elute the enzyme from the Ly6 domain. When LPL was in complex with the acidic peptide corresponding to the N-terminal domain of GPIHBP1, the enzyme retained its affinity for the Ly6 domain. Furthermore, LPL that was bound to the N-terminal domain interacted with lipoproteins, whereas LPL bound to the Ly6 domain did not. In summary, our data suggest that the two domains of GPIHBP1 interact independently with LPL and that the functionality of LPL depends on its localization on GPIHBP1. 相似文献
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Padmaja L. Ghospurkar Timothy M. Wilson Amber L. Severson Sarah J. Klein Sakina K. Khaku André P. Walther Stuart J. Haring 《Genetics》2015,199(3):711-727
In response to DNA damage, two general but fundamental processes occur in the cell: (1) a DNA lesion is recognized and repaired, and (2) concomitantly, the cell halts the cell cycle to provide a window of opportunity for repair to occur. An essential factor for a proper DNA-damage response is the heterotrimeric protein complex Replication Protein A (RPA). Of particular interest is hyperphosphorylation of the 32-kDa subunit, called RPA2, on its serine/threonine-rich amino (N) terminus following DNA damage in human cells. The unstructured N-terminus is often referred to as the phosphorylation domain and is conserved among eukaryotic RPA2 subunits, including Rfa2 in Saccharomyces cerevisiae. An aspartic acid/alanine-scanning and genetic interaction approach was utilized to delineate the importance of this domain in budding yeast. It was determined that the Rfa2 N-terminus is important for a proper DNA-damage response in yeast, although its phosphorylation is not required. Subregions of the Rfa2 N-terminus important for the DNA-damage response were also identified. Finally, an Rfa2 N-terminal hyperphosphorylation-mimetic mutant behaves similarly to another Rfa1 mutant (rfa1-t11) with respect to genetic interactions, DNA-damage sensitivity, and checkpoint adaptation. Our data indicate that post-translational modification of the Rfa2 N-terminus is not required for cells to deal with “repairable” DNA damage; however, post-translational modification of this domain might influence whether cells proceed into M-phase in the continued presence of unrepaired DNA lesions as a “last-resort” mechanism for cell survival. 相似文献
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The Carboxyl-Terminal Region of the Human Papillomavirus Type 16 E1 Protein Determines E2 Protein Specificity during DNA Replication 总被引:2,自引:1,他引:1
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Nianxiang Zou Jen-Sing Liu Shu-Ru Kuo Thomas R. Broker Louise T. Chow 《Journal of virology》1998,72(4):3436-3441
The mechanism of DNA replication is conserved among papillomaviruses. The virus-encoded E1 and E2 proteins collaborate to target the origin and recruit host DNA replication proteins. Expression vectors of E1 and E2 proteins support homologous and heterologous papillomaviral origin replication in transiently transfected cells. Viral proteins from different genotypes can also collaborate, albeit with different efficiencies, indicating a certain degree of specificity in E1-E2 interactions. We report that, in the assays of our study, the human papillomavirus type 11 (HPV-11) E1 protein functioned with the HPV-16 E2 protein, whereas the HPV-16 E1 protein exhibited no detectable activity with the HPV-11 E2 protein. Taking advantage of this distinction, we used chimeric E1 proteins to delineate the E1 protein domains responsible for this specificity. Hybrids containing HPV-16 E1 amino-terminal residues up to residue 365 efficiently replicated either viral origin in the presence of either E2 protein. The reciprocal hybrids containing amino-terminal HPV-11 sequences exhibited a high activity with HPV-16 E2 but no activity with HPV-11 E2. Reciprocal hybrid proteins with the carboxyl-terminal 44 residues from either E1 had an intermediate property, but both collaborated more efficiently with HPV-16 E2 than with HPV-11 E2. In contrast, chimeras with a junction in the putative ATPase domain showed little or no activity with either E2 protein. We conclude that the E1 protein consists of distinct structural and functional domains, with the carboxyl-terminal 284 residues of the HPV-16 E1 protein being the primary determinant for E2 specificity during replication, and that chimeric exchanges in or bordering the ATPase domain inactivate the protein. 相似文献
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Characterization of Episomal Replication of Bovine Papillomavirus Type 1 DNA in Long-Term Virion-Infected Saccharomyces Cerevisiae Culture
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Tu Quanmei Feng Weixu Chen Zhuo Li Qijia Zhao Yu Chen Jun Jiang Pengfei Xue Xiangyang Zhang Lifang Zhao Kong-Nan 《中国病毒学》2021,36(6):1492-1502
Virologica Sinica - We have previously reported that bovine papillomavirus type 1 (BPV-1) DNA can replicate its genome and produce infectious virus-like particles in short term virion-infected S.... 相似文献
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Distinct Roles of RAG1 and RAG2 in Binding the V(D)J Recombination Signal Sequences 总被引:15,自引:10,他引:5
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The RAG1 and RAG2 proteins initiate V(D)J recombination by introducing double-strand breaks at the border between a recombination signal sequence (RSS) and a coding segment. To understand the distinct functions of RAG1 and RAG2 in signal recognition, we have compared the DNA binding activities of RAG1 alone and RAG1 plus RAG2 by gel retardation and footprinting analyses. RAG1 exhibits only a three- to fivefold preference for binding DNA containing an RSS over random sequence DNA. Although direct binding of RAG2 by itself was not detected, the presence of both RAG1 and RAG2 results in the formation of a RAG1-RAG2-DNA complex which is more stable and more specific than the RAG1-DNA complex and is active in V(D)J cleavage. These results suggest that biologically effective discrimination between an RSS and nonspecific sequences requires both RAG1 and RAG2. Unlike the binding of RAG1 plus RAG2, RAG1 can bind to DNA in the absence of a divalent metal ion and does not require the presence of coding flank sequence. Footprinting of the RAG1-RAG2 complex with 1,10-phenanthroline-copper and dimethyl sulfate protection reveal that both the heptamer and the nonamer are involved. The nonamer is protected, with extensive protein contacts within the minor groove. Conversely, the heptamer is rendered more accessible to chemical attack, suggesting that binding of RAG1 plus RAG2 distorts the DNA near the coding/signal border. 相似文献
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In this study, we present data that support the presence of two distinct calmodulin binding sites within the angiotensin II receptor (AT1A), at juxtamembrane regions of the N-terminus of the third intracellular loop (i3, amino acids 214–231) and carboxyl tail of the receptor (ct, 302–317). We used bioluminescence resonance energy transfer assays to document interactions of calmodulin with the AT1A holo-receptor and GST-fusion protein pull-downs to demonstrate that i3 and ct interact with calmodulin in a Ca2+-dependent fashion. The former is a 1–12 motif and the latter belongs to 1-5-10 calmodulin binding motif. The apparent Kd of calmodulin for i3 is 177.0±9.1 nM, and for ct is 79.4±7.9 nM as assessed by dansyl-calmodulin fluorescence. Replacement of the tryptophan (W219) for alanine in i3, and phenylalanine (F309 or F313) for alanine in ct reduced their binding affinities for calmodulin, as predicted by computer docking simulations. Exogenously applied calmodulin attenuated interactions between G protein βγ subunits and i3 and ct, somewhat more so for ct than i3. Mutations W219A, F309A, and F313A did not alter Gβγ binding, but reduced the ability of calmodulin to compete with Gβγ, suggesting that calmodulin and Gβγ have overlapping, but not identical, binding requirements for i3 and ct. Calmodulin interference with the Gβγ binding to i3 and ct regions of the AT1A receptor strongly suggests that calmodulin plays critical roles in regulating Gβγ-dependent signaling of the receptor. 相似文献
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Y Watanabe T Kobayashi H Yamamoto H Hoshida R Akada F Inagaki Y Ohsumi NN Noda 《The Journal of biological chemistry》2012,287(38):31681-31690
Autophagy is an intracellular degradation system by which cytoplasmic materials are enclosed by an autophagosome and delivered to a lysosome/vacuole. Atg18 plays a critical role in autophagosome formation as a complex with Atg2 and phosphatidylinositol 3-phosphate (PtdIns(3)P). However, little is known about the structure of Atg18 and its recognition mode of Atg2 or PtdIns(3)P. Here, we report the crystal structure of Kluyveromyces marxianus Hsv2, an Atg18 paralog, at 2.6 Å resolution. The structure reveals a seven-bladed β-propeller without circular permutation. Mutational analyses of Atg18 based on the K. marxianus Hsv2 structure suggested that Atg18 has two phosphoinositide-binding sites at blades 5 and 6, whereas the Atg2-binding region is located at blade 2. Point mutations in the loops of blade 2 specifically abrogated autophagy without affecting another Atg18 function, the regulation of vacuolar morphology at the vacuolar membrane. This architecture enables Atg18 to form a complex with Atg2 and PtdIns(3)P in parallel, thereby functioning in the formation of autophagosomes at autophagic membranes. 相似文献
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《Journal of molecular biology》2022,434(17):167646
The effect of perturbation at the allosteric site was investigated through several replicas of molecular dynamics (MD) simulations conducted on bacterial phosphofructokinase (SaPFK). In our previous work, an alternative binding site was estimated to be allosteric in addition to the experimentally reported one. To highlight the effect of both allosteric sites on receptor’s dynamics, MD runs were carried out on apo forms with and without perturbation. Perturbation was achieved via incorporating multiple bond restraints for residue pairs located at the allosteric site. Restraints applied to the predicted site caused one dimer to stiffen, whereas an increase in mobility was detected in the same dimer when the experimentally resolved site was restrained. Fluctuations in Cα-Cα distances which is used to disclose residues with high potential of communication indicated a marked increase in signal transmission within each dimer as the receptor switched to a restrained state. Cross-correlation of positional fluctuations indicated an overall decrease in the magnitude of both positive and negative correlations when restraints were employed on the predicted allosteric site whereas an exact opposite effect was observed for the reported site. Finally, mutual correspondence between positional fluctuations noticeably increased with restraints on predicted allosteric site, whereas an opposite effect was observed for restraints applied on experimentally reported one. In view of these findings, it is clear that the perturbation of either one of two allosteric sites effected the dynamics of the receptor with a distinct and contrasting character. 相似文献