TGMV replication protein AL1 preferentially binds to single‐stranded DNA from the common region

Thömmes, Pia; Osman, T. A. M.; Hayes, Robert J.; Buck, K. W.

doi:10.1016/0014-5793(93)80044-u

Cited by 38 publications

(22 citation statements)

References 21 publications

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“…Although ACMV AC1 binding has not yet been investigated, the simplest interpretation of these data is that the N-terminal portion of AC1 interacts with sequences within domain A to suppress AC1 expression. The idea is consistent with reports describing the specific binding of ALl of TGMV and bean golden mosaic virus to sequences within the common region equivalent to domain A (Fontes et al, 1992(Fontes et al, , 1994aTh6mmes et al, 1993). A more detailed analysis of the TGMV ALl binding site and its involvement in transcriptional repression has identified a 13 bp element as a high affinity binding site, located nine nucleotides upstream of the ALl transcription start point (Fontes et aI., 1994b;Eagle et al, 1994).…”

Section: Discussionsupporting

confidence: 91%

Regulation of African cassava mosaic virus complementary-sense gene expression by N-terminal sequences of the replication-associated protein AC1

Hong

Stanley

1995

Journal of General Virology

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Section: Discussionsupporting

confidence: 91%

Regulation of African cassava mosaic virus complementary-sense gene expression by N-terminal sequences of the replication-associated protein AC1

Hong

Stanley

1995

Journal of General Virology

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“…lanes 2 to 4 and lanes 6 to 8), indicating that AL1 binds to ssDNA in a sequence-nonspecific manner. Our result contrasts with an earlier study (70) that concluded that AL1 binds preferentially to a ssDNA sequence corresponding to TGMV common region based on binding to larger probes containing the entire common region (CR in Fig. S2A in the supplemental material).…”

Section: Vol 85 2011 Novel Motif Conserved Across Geminivirus Rep Pcontrasting

confidence: 99%

Functional Analysis of a Novel Motif Conserved across Geminivirus Rep Proteins

Nash

Dallas

Reyes

et al. 2011

J Virol

109

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Members of the Geminiviridae have single-stranded DNA genomes that replicate in nuclei of infected plant cells. All geminiviruses encode a conserved protein (Rep) that catalyzes initiation of rolling-circle replication. Earlier studies showed that three conserved motifs-motifs I, II, and III-in the N termini of geminivirus Rep proteins are essential for function. In this study, we identified a fourth sequence, designated GRS (geminivirus Rep sequence), in the Rep N terminus that displays high amino acid sequence conservation across all geminivirus genera. Using the Rep protein of Tomato golden mosaic virus (TGMV AL1), we show that GRS mutants are not infectious in plants and do not support viral genome replication in tobacco protoplasts. GRS mutants are competent for protein-protein interactions and for both double-and single-stranded DNA binding, indicating that the mutations did not impair its global conformation. In contrast, GRS mutants are unable to specifically cleave single-stranded DNA, which is required to initiate rolling-circle replication. Interestingly, the Rep proteins of phytoplasmal and algal plasmids also contain GRS-related sequences. Modeling of the TGMV AL1 N terminus suggested that GRS mutations alter the relative positioning of motif II, which coordinates metal ions, and motif III, which contains the tyrosine involved in DNA cleavage. Together, these results established that the GRS is a conserved, essential motif characteristic of an ancient lineage of rolling-circle initiators and support the idea that geminiviruses may have evolved from plasmids associated with phytoplasma or algae.Geminiviruses are plant viruses with small, circular DNA genomes and twin icosahedral capsids (58). They constitute a large family that is divided into the begomovirus, mastrevirus, curtovirus, and topocuvirus genera based on genome arrangement, insect vector, and host range (68). Geminiviruses amplify their single-stranded (ss) genomes in the nuclei of infected cells using a combination of rolling-circle and recombination-mediated replication (reviewed in references 27, 31, and 35). All geminiviruses encode a conserved protein designated Rep (also known as AL1, AC1, C1, L1, or C1:C2), which mediates initiation of viral replication but does not act as a DNA polymerase (33,42,51). Instead, geminiviruses depend on host replication machinery to copy their genomes (28,31,32).Rep is the only geminivirus protein that is essential for viral replication (18, 56). It is a multifunctional protein that mediates virus-specific recognition of its cognate origin (22) and transcriptional repression (17, 69). Rep initiates and terminates viral DNA synthesis (33, 42, 51) and induces the accumulation of host replication factors in infected cells (48). Rep binds specifically to double-stranded DNA (dsDNA) at a repeated sequence in the 5Ј intergenic region of the viral genome (22, 67), cleaves and ligates DNA within an invariant sequence in a hairpin loop of the plus-strand origin (42, 51), and acts as a DNA helicase to unwind vi...

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“…The radiolabeled band has an apparent molecular mass of 23 kDa, which is slightly larger than the protein itself, a condition which is most likely caused by the addition of oligonucleotides to the polypeptide upon cross-linking. Similar size shifts were observed for other DNA binding proteins, including the large subunit of dRP-A (14) and the tomato golden mosaic virus protein AL1 (21).…”

Section: Purification Of Dm Mtssbsupporting

confidence: 70%

Mitochondrial Single-stranded DNA-binding Protein from Drosophila Embryos

Thömmes

Farr

Marton

et al. 1995

Journal of Biological Chemistry

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Using a stringent purification procedure on singlestranded DNA cellulose, we have isolated the mitochondrial single-stranded DNA-binding protein from Drosophila melanogaster embryos. Its identity is demonstrated by amino-terminal sequencing of the homogeneous protein and by its localization to a mitochondrial protein fraction. The mitochondrial protein is immunologically and biochemically distinct from the previously characterized nuclear replication protein A from Drosophila (Mitsis, P. G., Kowalczykowski, S. C., and Lehman, I. R. Many of the processes involved in DNA metabolism including DNA replication, recombination, and repair, generate intermediates containing single-stranded regions of DNA. These regions are stabilized and kept accessible for the various catalytic processes by the binding of single-stranded DNA-binding proteins (SSBs).1 Prokaryotic SSBs (e.g. Escherichia coli (Eco) SSB and bacteriophage T4 gene 32 protein) are generally small proteins which bind to single-stranded DNA (ssDNA) with high affinity. They show high specificity for ssDNA over doublestranded DNA (dsDNA) and RNA, but display little sequence specificity (reviewed in Refs. 1-3). Although they do not exhibit direct catalytic function, they stimulate DNA replication in vitro. Mitochondrial DNA replication is independent from chromosomal DNA replication and is carried out largely with specific mitochondrial replication proteins including the mitochondrial DNA polymerase (pol ␥) and an SSB (mtSSB) distinct from the nuclear SSB, replication protein A (RP-A). mtSSB appears to serve an important function during mtDNA replication, by stabilizing the displaced ssDNA that is the template for lagging DNA strand synthesis (4). mtSSBs have been isolated from several species including rat (4, 5), Xenopus laevis (6), and yeast (7). These proteins consist of a single small (13-16 kDa) polypeptide, which shows a high degree of similarity to Eco SSB in its primary structure (7,8). Although all the functions of mtSSB in mtDNA metabolism have not been defined, it is critical for replication, because deletion of the yeast protein (RIM1) causes loss of mitochondrial DNA (7). Consistent with a role in mtDNA replication, interactions between mtSSB and other mitochondrial replication proteins have been observed. In vitro studies indicate that under some conditions, the rat and X. laevis mtSSBs stimulate partially purified forms of mitochondrial DNA polymerase (9, 10), and a putative human mtSSB stimulates human pol ␥ (11). In addition, genetic evidence from yeast suggests an interaction between RIM1 and the mtDNA helicase, PIF1 (7).We have purified a single-stranded DNA-binding protein from Drosophila embryos (hereafter called Dm mtSSB) to near homogeneity. Its physical and biochemical properties demonstrate that it is distinct from the nuclear SSB, dRP-A, but has a high degree of similarity to Eco SSB and to eukaryotic mtSSBs. Further, its functional interaction with the near-homogeneous mitochondrial DNA polymerase from Drosophila melanogaster embryos (1...

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TGMV replication protein AL1 preferentially binds to single‐stranded DNA from the common region

Cited by 38 publications

References 21 publications

Regulation of African cassava mosaic virus complementary-sense gene expression by N-terminal sequences of the replication-associated protein AC1

Regulation of African cassava mosaic virus complementary-sense gene expression by N-terminal sequences of the replication-associated protein AC1

Functional Analysis of a Novel Motif Conserved across Geminivirus Rep Proteins

Mitochondrial Single-stranded DNA-binding Protein from Drosophila Embryos

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