The first zinc-binding domain of UvrA is not essential for UvrABC-mediated DNA excision repair

Visse, Robert; Ruijter, M de; Ubbink, Marcellus; Brandsma, Jourica A.; Putte, Pieter van de

doi:10.1016/0921-8777(93)90009-6

Cited by 24 publications

(23 citation statements)

References 36 publications

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“…A C 4 -type zinc finger is present within each insertion segment. Previously, it was thought that UvrA interacts with DNA via the zinc finger at the C-terminus of the protein [23,24]. Recently, we observed increased DNA binding when amino acids within this zinc finger were deleted [25].…”

Section: Introductionmentioning

confidence: 88%

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Cooperative damage recognition by UvrA and UvrB: Identification of UvrA residues that mediate DNA binding

et al. 2008

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Nucleotide excision repair (NER) is responsible for the recognition and removal of numerous structurally unrelated DNA lesions. In prokaryotes, the proteins UvrA, UvrB and UvrC orchestrate the recognition and excision of aberrant lesions from DNA. Despite the progress we have made in understanding the NER pathway it remains unclear how the UvrA dimer interacts with DNA to facilitate DNA damage recognition. The purpose of this study was to define amino acids residues in UvrA that provide binding energy to DNA. Based on conservation among ~300 UvrA sequences and 3D-modeling, two positively charged residues, Lys680 and Arg691, were predicted to be important for DNA binding. Mutagenesis and biochemical analysis of Bacillus caldontenax UvrA variant proteins containing site directed mutations at these residues demonstrate that Lys680 and Arg691 make a significant contribution toward the DNA binding affinity of UvrA. Replacing these side chains with alanine or negatively charged residues decreased UvrA binding 3-37-fold. Survival studies indicated that these mutant proteins complemented a WP2 uvrA − strain of bacteria 10% to 100% of WT UvrA levels. Further analysis by DNase I footprinting of the double UvrA mutant revealed that the UvrA DNA binding defects caused a slower rate of transfer of DNA to UvrB. Consequently, the mutants initiated the oligonucleotide incision assay nearly as well as WT UvrA thus explaining the observed mild phenotype in the survival assay. Based on our findings we propose a model of how UvrA binds to DNA.

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Section: Introductionmentioning

confidence: 88%

“…Historically, it was thought that UvrA interacted with DNA via its zinc finger in the C-terminus of the protein [23,24]. However, we have recently shown that this zinc finger is not required for DNA binding by UvrA [25].…”

Section: Selection Of the Site Directed Mutagenesis Targetsmentioning

confidence: 99%

Cooperative damage recognition by UvrA and UvrB: Identification of UvrA residues that mediate DNA binding

et al. 2008

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“…Interestingly, a similarly positioned 14-aa region containing a Ser-Pro dipeptide is adjacent to the C-terminal Zn 2ϩ binding Cys of the proven Zn 2ϩ fingers in the GAL4 and LAC9 proteins (49,(70)(71)(72). The arterivirus MBD may adopt a unique multinuclear organization and bind four Zn 2؉ s. The combination of a putative MBD and a Hel domain in one protein, as in EAV nsp10, is found in a number of viral and cellular proteins (18,27,30,33,40,45,67), including the replicases of related nidoviruses (12,23,28,52). At the primary structure level, the MBDs of all these proteins are dissimilar from the arterivirus one.…”

Section: Discussionmentioning

confidence: 99%

The Predicted Metal-Binding Region of the Arterivirus Helicase Protein Is Involved in Subgenomic mRNA Synthesis, Genome Replication, and Virion Biogenesis

Dinten

Tol

Gorbalenya

et al. 2000

J Virol

100

131

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Equine arteritis virus (EAV), the prototype Arterivirus, is a positive-stranded RNA virus that expresses its replicase in the form of two large polyproteins of 1,727 and 3,175 amino acids. The functional replicase subunits (nonstructural proteins), which drive EAV genome replication and subgenomic mRNA transcription, are generated by extensive proteolytic processing. Subgenomic mRNA transcription involves an unusual discontinuous step and generates the mRNAs for structural protein expression. Previously, the phenotype of mutant EAV030F, which carries a single replicase point mutation (Ser-24293Pro), had implicated the nsp10 replicase subunit (51 kDa) in viral RNA synthesis, and in particular in subgenomic mRNA transcription. nsp10 contains an N-terminal (putative) metal-binding domain (MBD), located just upstream of the Ser-24293Pro mutation, and a helicase activity in its C-terminal part. We have now analyzed the N-terminal domain of nsp10 in considerable detail. A total of 38 mutants, most of them carrying specific single point mutations, were tested in the context of an EAV infectious cDNA clone. Variable effects on viral genome replication and subgenomic mRNA transcription were observed. In general, our results indicated that the MBD region, and in particular a set of 13 conserved Cys and His residues that are assumed to be involved in zinc binding, is essential for viral RNA synthesis. On the basis of these data and comparative sequence analyses, we postulate that the MBD may employ a rather unusual mode of zinc binding that could result in the association of up to four zinc cations with this domain. The region containing residue Ser-2429 may play the role of "hinge spacer," which connects the MBD to the rest of nsp10. Several mutations in this region specifically affected subgenomic mRNA synthesis. Furthermore, one of the MBD mutants was replication and transcription competent but did not produce infectious progeny virus. This suggests that nsp10 is involved in an as yet unidentified step of virion biogenesis.Equine arteritis virus (EAV) (17) is the prototype of the Arteriviridae, a recently established family of positive-stranded, enveloped RNA viruses (for reviews see references 47 and 53). The other three members of the Arteriviridae are Lactate dehydrogenase-elevating virus (LDV), Porcine reproductive and respiratory syndrome virus (PRRSV), and Simian hemorrhagic fever virus (SHFV). Based on their presumed common ancestry (7), the arteriviruses have been united with the coronaviruses in the order of the Nidovirales. Important common properties of nidoviruses (Fig. 1) are the presence of a unique array of conserved domains in the viral replicase, the use of ribosomal frameshifting and proteolytic processing to regulate replicase gene expression, and the use of discontinuous transcription to generate a nested set of subgenomic (sg) mRNAs for structural protein expression (for reviews see references 15, 35, and 53).The EAV nonstructural proteins are generated by proteolytic processing of two large replica...

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“…The arterivirus helicase is amino terminally linked to a putative Zn finger structure (6). This combination of a Zn finger structure with a helicase domain is also found in the related coronavirus helicases (7,17,23) and a number of cellular and viral helicases (9,25,34,39,58). Recently, genetic evidence was obtained to show that both the Zn finger itself and the region connecting the Zn finger to the carboxyl proximal part of nsp10 ("hinge spacer") are critically involved in different processes of the EAV life cycle, including genome replication, mRNA transcription, and possibly also virion biogenesis (53,55,57).…”

mentioning

confidence: 95%

Biochemical Characterization of the Equine Arteritis Virus Helicase Suggests a Close Functional Relationship between Arterivirus and Coronavirus Helicases

Seybert

Dinten

Snijder

et al. 2000

J Virol

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The arterivirus equine arteritis virus nonstructural protein 10 (nsp10) has previously been predicted to contain a Zn finger structure linked to a superfamily 1 (SF1) helicase domain. A recombinant form of nsp10, MBP-nsp10, was produced in Escherichia coli as a fusion protein with the maltose-binding protein. The protein was partially purified by affinity chromatography and shown to have ATPase activity that was strongly stimulated by poly(dT), poly(U), and poly(dA) but not by poly(G). The protein also had both RNA and DNA duplex-unwinding activities that required the presence of 5 single-stranded regions on the partial-duplex substrates, indicating a 5-to-3 polarity in the unwinding reaction. Results of this study suggest a close functional relationship between the arterivirus nsp10 and the coronavirus helicase, for which NTPase and duplex-unwinding activities were recently demonstrated. In a number of biochemical properties, both arterivirus and coronavirus SF1 helicases differ significantly from the previously characterized RNA virus SF1 and SF2 enzymes. Thus, the combined data strongly support the idea that nidovirus helicases may represent a separate group of RNA virus-encoded helicases with distinct properties.Equine arteritis virus (EAV) is the prototype of the Arteriviridae, a family of positive-stranded, enveloped RNA viruses which also includes Lactate dehydrogenase-elevating virus, Porcine reproductive and respiratory syndrome virus, and Simian haemorrhagic fever virus (for a review, see 47). A common ancestry of the Arteriviridae and Coronaviridae seems probable (6), and, consequently, the two families have been united in the order Nidovirales (3). The phylogenetic relationship between arteri-and coronaviruses is most evident from the organization and expression of their replicase genes. Thus, for example, both arteri-and coronaviruses (i) encode a very similar array of functional domains in their replicase genes, (ii) use ribosomal frameshifting to express key replicative functions, (iii) control the activity of the individual subunits of the viral replication and transcription machinery by extensive proteolytic processing of large protein precursors, and (iv) use a discontinuous transcription mechanism to produce a nested set of subgenomic (sg) mRNAs for structural gene expression (3,8).The EAV replicase gene comprises the 5Ј-terminal threefourths of the 12.7-kb genome and is composed of two open reading frames (ORFs), ORF1a and ORF1b (6). The upstream ORF1a encodes the ORF1a protein (187 kDa), and ORF1a and ORF1b together encode the ORF1ab protein (345 kDa). Expression of the ORF1b-encoded part of the ORFlab protein involves a ribosomal frameshift in the ORF1a-1b overlap region during translation of the genomic RNA (6). The primary translation products, which are also called replicase polyproteins, are extensively processed by three virus-encoded proteinases to produce 12 mature proteins (nonstructural protein 1 [nsp1] to nsp12), as well as multiple processing intermediates (for a recent review, s...

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The first zinc-binding domain of UvrA is not essential for UvrABC-mediated DNA excision repair

Cited by 24 publications

References 36 publications

Cooperative damage recognition by UvrA and UvrB: Identification of UvrA residues that mediate DNA binding

Cooperative damage recognition by UvrA and UvrB: Identification of UvrA residues that mediate DNA binding

The Predicted Metal-Binding Region of the Arterivirus Helicase Protein Is Involved in Subgenomic mRNA Synthesis, Genome Replication, and Virion Biogenesis

Biochemical Characterization of the Equine Arteritis Virus Helicase Suggests a Close Functional Relationship between Arterivirus and Coronavirus Helicases

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