Conformational Change in the Herpes Simplex Single-Strand Binding Protein Induced by DNA

Dudas, Kathleen C.; Scouten, Sarah K.; Ruyechan, William T.

doi:10.1006/bbrc.2001.5766

Cited by 5 publications

(8 citation statements)

References 45 publications

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“…33 For completeness, this was repeated here and the results (Figure 2(b) and data not shown) were in excellent accord with those of the previous study. In brief, the addition of Mg 2C did not change the pattern of proteolysis significantly, but the pattern is different in the presence or in the absence of ssDNA, irrespective of the presence of Mg 2C .…”

Section: Resultssupporting

confidence: 86%

“…Partial proteolysis of ICP8 assembled into nucleoprotein filaments reveals Mg 2C -dependent conformational changes Partial proteolysis of ICP8 in the presence and in the absence of ssDNA has been described by Dudas and co-workers, 33 who observed conformational changes depending on whether ICP8 was free or bound to ssDNA, and the region affected was localized between amino acid residues 293 and 806, which contains the ssDNA-binding region. In their study, the influence of Mg 2C on the proteolysis was not investigated.…”

Section: Resultsmentioning

confidence: 81%

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Visualization of the Annealing of Complementary Single-stranded DNA Catalyzed by the Herpes Simplex Virus Type 1 ICP8 SSB/Recombinase

Makhov

Griffith

2006

Journal of Molecular Biology

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Section: Resultssupporting

confidence: 86%

Section: Resultsmentioning

confidence: 81%

Visualization of the Annealing of Complementary Single-stranded DNA Catalyzed by the Herpes Simplex Virus Type 1 ICP8 SSB/Recombinase

Makhov

Griffith

2006

Journal of Molecular Biology

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“…An alternative possibility is that ICP8 may undergo conformational alterations as a result of DNA binding. The reported quenching of ICP8 tryptophan fluorescence, changes in protease cleavage patterns, and changes in antibody quenching of fluorescein-5-maleimide-modified ICP8 in the presence of single-stranded DNA are consistent with a DNA-induced conformational change (18,70). Changes in protein conformation upon DNA binding have been documented for a number of proteins (reviewed in reference 73), including the HSV polymerase (86), the VP16 interacting protein Oct-1 (58), and the cellular SSB RP-A (25).…”

Section: Discussionsupporting

confidence: 58%

“…Another obvious issue to consider is whether and how these antigenic changes are related to ICP8 activity. Using sensitive biochemical assays, other studies have shown conformational changes in purified ICP8 upon binding to DNA (18,70). Our genetic analysis showed an association of DNA binding with the presence of the 39S epitope.…”

Section: Discussionmentioning

confidence: 99%

“…Second, within cells infected with an ICP27 null mutant virus, immunofluorescence assays revealed a partial defect in ICP8 localization to viral replication structures and a corresponding difference in ICP8 reactivity with the conformation-specific 39S monoclonal antibody (MAb) (15). Finally, more recent biochemical studies on purified ICP8 have demonstrated an apparent conformational change in ICP8 upon binding to single-stranded DNA in vitro (18).…”

mentioning

confidence: 99%

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Conformational Changes in the Herpes Simplex Virus ICP8 DNA-Binding Protein Coincident with Assembly in Viral Replication Structures

Uprichard

Knipe

2003

J Virol

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The herpes simplex virus (HSV) single-stranded DNA-binding protein, ICP8, is required for viral DNA synthesis. Before viral DNA replication, ICP8 colocalizes with other replication proteins at small punctate foci called prereplicative sites. With the onset of viral genome amplification, these proteins become redistributed into large globular replication compartments. Here we present the results of immunocytochemical and biochemical analysis of ICP8 showing that various antibodies recognize distinct forms of ICP8. Using these ICP8-specific antibodies as probes for ICP8 structure, we detected a time-dependent appearance and disappearance of ICP8 epitopes in immunoprecipitation assays. Immunofluorescence staining of ICP8 in cells infected with different HSV mutant viruses as well as cells transfected with a limited number of viral genes demonstrated that these and other antigenic changes occur coincident with ICP8 assembly at intranuclear replication structures. Genetic analysis has revealed a correlation between the ability of various ICP8 mutant proteins to form the 39S epitope and their ability to bind to DNA. These results support the hypothesis that ICP8 undergoes a conformational change upon binding to other HSV proteins and/or to DNA coincident with assembly into viral DNA replication structures.Changes in protein conformation can be critical to polypeptide function. Therefore, a complete understanding of how some proteins are regulated involves identifying changes in their tertiary structure. For example, structural changes in many transcription factors (e.g., OxyR, AP-1, Sp-1, NF-B, and p53) (reviewed in references 28, 29, 34, 71, 74, and 91), the Escherichia coli replication protein dnaB (2, 3, 35), adenovirus 72K single-stranded DNA-binding protein (SSB) (17), bacteriophage T4 gene 32 SSB (85), and the eukaryotic SSB replication protein A (RP-A) (6, 25) are functionally related to the activity of these proteins. Changes in protein structure can be regulated by a variety of means, including binding to DNA (reviewed in references 25 and 73), interactions with ions (84, 88), interactions with other proteins (19,47,66), posttranslational modifications (46, 49; reviewed in reference 36), or even environmental conditions, such as redox potential (reviewed in references 5 and 13).We have studied herpes simplex virus (HSV) DNA replication as a model system for the localization, maturation, and ordered assembly of protein complexes within the cell. HSV encodes seven proteins that are necessary for viral origin-dependent DNA synthesis (12,39,75,87,90). These include the SSB (ICP8), as well as a polymerase (U L 30), its processivity factor (U L 42), an origin-binding protein (U L 9), and three proteins that form the viral helicase-primase complex (U L 5, U L 8, and U L 52). ICP8 serves several functions during viral DNA synthesis (67). It functions as an SSB to stabilize displaced single-stranded DNA strands during HSV DNA replication and also stimulates the helicase activity of U L 9 during initiation and that of ...

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Structural and functional analysis of the baculovirus single-stranded DNA-binding protein LEF-3

2006

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The single-stranded DNA-binding protein LEF-3 of Autographa californica multinucleocapsid nucleopolyhedrovirus consists of 385 amino acid residues, forms oligomers, and promotes Mg2+-independent unwinding of DNA duplexes and annealing of complementary DNA strands. Partial proteolysis revealed that the DNA-binding domain of LEF-3 is located within a central region (residues 28 to 326) that is relatively resistant to proteolysis. In contrast, the N-terminus (27 residues) and C-terminal portion (59 residues) are not involved in interaction with DNA and are readily accessible to proteolytic digestion. Circular dichroism analyses showed that LEF-3 is a folded protein with an estimated alpha-helix content of more than 40%, but it is structurally unstable and undergoes unfolding in aqueous solutions at temperatures near 50 degrees C. Unfolding eliminated the LEF-3 domains that are resistant to proteolysis and randomized the digestion pattern by trypsin. The structural transition was irreversible and was accompanied by the generation of high molecular weight (MW) complexes. The thermal treatment inhibited DNA-binding and unwinding activity of LEF-3 but markedly stimulated its annealing activity. We propose that the shift in LEF-3 activities resulted from the generation of the high MW protein complexes, that specifically stimulate the annealing of complementary DNA strands by providing multiple DNA-binding sites and bringing into close proximity the interacting strands. The unfolded LEF-3 was active in a strand exchange reaction suggesting that it could be involved in the production of recombination intermediates.

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Conformational Change in the Herpes Simplex Single-Strand Binding Protein Induced by DNA

Cited by 5 publications

References 45 publications

Visualization of the Annealing of Complementary Single-stranded DNA Catalyzed by the Herpes Simplex Virus Type 1 ICP8 SSB/Recombinase

Visualization of the Annealing of Complementary Single-stranded DNA Catalyzed by the Herpes Simplex Virus Type 1 ICP8 SSB/Recombinase

Conformational Changes in the Herpes Simplex Virus ICP8 DNA-Binding Protein Coincident with Assembly in Viral Replication Structures

Structural and functional analysis of the baculovirus single-stranded DNA-binding protein LEF-3

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