scholarly journals Unlimited cooperativity of Betatectivirus SSB, a novel DNA binding protein related to an atypical group of SSBs from protein-primed replicating bacterial viruses

2021 ◽  
Author(s):  
Ana Lechuga ◽  
Darius Kazlauskas ◽  
Margarita Salas ◽  
Modesto Redrejo Rodriguez
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Electrostatic Interactions ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Structural Domain ◽  
First Occurrence ◽  
Domains Of Life ◽  
Ob Fold ◽  
Specific Stimulation

Bam35 and related betatectiviruses are tail-less bacteriophages that prey on members of the Bacillus cereus group. These temperate viruses replicate their linear genome by a protein-primed mechanism. In this work, we have identified and characterized the product of the viral ORF2 as a single-stranded DNA binding protein (hereafter B35SSB). B35SSB binds ssDNA with great preference over dsDNA or RNA in a sequence-independent, highly cooperative manner that results in a non-specific stimulation of DNA replication. We have also identified several aromatic and basic residues, involved in base-stacking and electrostatic interactions, respectively, that are required for effective protein-ssDNA interaction. Although SSBs are essential for DNA replication in all domains of life as well as many viruses, they are very diverse proteins. However, most SSBs share a common structural domain, named OB-fold. Protein-primed viruses could constitute an exception, as no OB-fold DNA binding protein has been reported. Based on databases searches as well as phylogenetic and structural analyses, we showed that B35SSB belongs to a novel and independent group of SSBs. This group contains proteins encoded by protein-primed viral genomes from unrelated viruses, spanning betatectiviruses and Φ29 and close podoviruses, and they share a conserved pattern of secondary structure. Sensitive searches and structural predictions indicate that B35SSB contains a conserved domain resembling a divergent OB-fold, which would constitute the first occurrence of an OB-fold-like domain in a protein-primed genome.

scholarly journals Unlimited Cooperativity of Betatectivirus SSB, a Novel DNA Binding Protein Related to an Atypical Group of SSBs From Protein-Primed Replicating Bacterial Viruses

2021 ◽  
Vol 12 ◽  
Author(s):  
Ana Lechuga ◽  
Darius Kazlauskas ◽  
Margarita Salas ◽  
Modesto Redrejo-Rodríguez
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Electrostatic Interactions ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Structural Domain ◽  
First Occurrence ◽  
Domains Of Life ◽  
Ob Fold ◽  
Specific Stimulation

Bam35 and related betatectiviruses are tail-less bacteriophages that prey on members of the Bacillus cereus group. These temperate viruses replicate their linear genome by a protein-primed mechanism. In this work, we have identified and characterized the product of the viral ORF2 as a single-stranded DNA binding protein (hereafter B35SSB). B35SSB binds ssDNA with great preference over dsDNA or RNA in a sequence-independent, highly cooperative manner that results in a non-specific stimulation of DNA replication. We have also identified several aromatic and basic residues, involved in base-stacking and electrostatic interactions, respectively, that are required for effective protein–ssDNA interaction. Although SSBs are essential for DNA replication in all domains of life as well as many viruses, they are very diverse proteins. However, most SSBs share a common structural domain, named OB-fold. Protein-primed viruses could constitute an exception, as no OB-fold DNA binding protein has been reported. Based on databases searches as well as phylogenetic and structural analyses, we showed that B35SSB belongs to a novel and independent group of SSBs. This group contains proteins encoded by protein-primed viral genomes from unrelated viruses, spanning betatectiviruses and Φ29 and close podoviruses, and they share a conserved pattern of secondary structure. Sensitive searches and structural predictions indicate that B35SSB contains a conserved domain resembling a divergent OB-fold, which would constitute the first occurrence of an OB-fold-like domain in a protein-primed genome.

scholarly journals Gene D5 product of bacteriophage T5: DNA-binding protein affecting DNA replication and late gene expression.

1979 ◽  
Vol 29 (1) ◽  
pp. 322-327 ◽  
Author(s):  
D J McCorquodale ◽  
J Gossling ◽  
R Benzinger ◽  
R Chesney ◽  
L Lawhorne ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Replication ◽  
Dna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Late Gene ◽  
Late Gene Expression ◽  
Bacteriophage T5

scholarly journals Nuclear localization of herpesvirus proteins: potential role for the cellular framework.

1983 ◽  
Vol 3 (3) ◽  
pp. 315-324 ◽  
Author(s):  
M P Quinlan ◽  
D M Knipe
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Capsid Protein ◽  
Cell Nucleus ◽  
Binding Protein ◽  
Cell Monolayer ◽  
Dna Binding Protein ◽  
Actual State ◽  
Viral Dna ◽  
Viral Dna Replication

Two herpes simplex virus proteins, the major capsid protein and the major DNA binding protein, are specifically localized to the nucleus of infected cells. We have found that the major proportion of these proteins is associated with the detergent-insoluble matrix or cytoskeletal framework of the infected cell from the time of their synthesis until they have matured to their final binding site in the cell nucleus. These results suggest that these two proteins may interact with or bind to the cellular cytoskeleton during or soon after their synthesis and throughout transport into the cell nucleus. In addition, the DNA binding protein remains associated with the nuclear skeleton at times when it is bound to viral DNA. Thus, viral DNA may also be attached to the nuclear framework. We have demonstrated that the DNA binding protein and the capsid protein exchange from the cytoplasmic framework to the nuclear framework, suggesting the direct movement of the proteins from one structure to the other. Inhibition of viral DNA replication enhanced the binding of the DNA binding protein to the cytoskeleton and increased the rate of exchange from the cytoplasmic framework to the nuclear framework, suggesting a functional relationship between these events. Inhibition of viral DNA replication resulted in decreased synthesis and transport of the capsid protein. We have been unable to detect any artificial binding of these proteins to the cytoskeleton when solubilized viral proteins were mixed with a cytoskeletal fraction or a cell monolayer. This suggested that the attachment of these proteins to the cytoskeleton represents the actual state of these proteins within the cell.

Nuclear localization of herpesvirus proteins: potential role for the cellular framework

1983 ◽  
Vol 3 (3) ◽  
pp. 315-324
Author(s):  
M P Quinlan ◽  
D M Knipe
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Capsid Protein ◽  
Cell Nucleus ◽  
Binding Protein ◽  
Cell Monolayer ◽  
Dna Binding Protein ◽  
Actual State ◽  
Viral Dna ◽  
Viral Dna Replication

Two herpes simplex virus proteins, the major capsid protein and the major DNA binding protein, are specifically localized to the nucleus of infected cells. We have found that the major proportion of these proteins is associated with the detergent-insoluble matrix or cytoskeletal framework of the infected cell from the time of their synthesis until they have matured to their final binding site in the cell nucleus. These results suggest that these two proteins may interact with or bind to the cellular cytoskeleton during or soon after their synthesis and throughout transport into the cell nucleus. In addition, the DNA binding protein remains associated with the nuclear skeleton at times when it is bound to viral DNA. Thus, viral DNA may also be attached to the nuclear framework. We have demonstrated that the DNA binding protein and the capsid protein exchange from the cytoplasmic framework to the nuclear framework, suggesting the direct movement of the proteins from one structure to the other. Inhibition of viral DNA replication enhanced the binding of the DNA binding protein to the cytoskeleton and increased the rate of exchange from the cytoplasmic framework to the nuclear framework, suggesting a functional relationship between these events. Inhibition of viral DNA replication resulted in decreased synthesis and transport of the capsid protein. We have been unable to detect any artificial binding of these proteins to the cytoskeleton when solubilized viral proteins were mixed with a cytoskeletal fraction or a cell monolayer. This suggested that the attachment of these proteins to the cytoskeleton represents the actual state of these proteins within the cell.

scholarly journals ClpX is Essential and Activated by Single Strand DNA Binding Protein in Mycobacteria

2020 ◽  
Author(s):  
Jemila C. Kester ◽  
Olga Kandror ◽  
Tatos Akopian ◽  
Michael R. Chase ◽  
Junhao Zhu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Dna Replication ◽  
Dna Binding ◽  
Drug Targets ◽  
Atp Hydrolysis ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Cellular Functions ◽  
Mycobacterial Growth ◽  
Dna Maintenance

The ClpP1P2 proteolytic complex is essential in Mycobacterium tuberculosis (Mtb). Proteolysis by ClpP1P2 requires an associated ATPase, either ClpX or ClpC1. Here, we seek to define the unique contributions of the ClpX ATPase to mycobacterial growth. We formally demonstrate that ClpX is essential for mycobacterial growth and to understand its essential functions, we identify ClpX-His-interacting proteins by pulldown and tandem mass spectrometry. We find an unexpected association between ClpX and proteins involved in DNA replication, and confirm a physical association between ClpX and the essential DNA maintenance protein Single-Stranded DNA Binding protein (SSB). Purified SSB is not degraded by ClpXP1P2; instead SSB enhances ATP hydrolysis by ClpX and degradation of the model substrate GFP-SsrA by ClpXP1P2. This activation of ClpX is mediated by the C-terminal tail of SSB that had been implicated in the activation of other ATPases associated with DNA replication. Consistent with the predicted interactions, depletion of clpX transcript perturbs DNA replication. These data reveal that ClpX participates in DNA replication and identify the first activator of ClpX in mycobacteria. IMPORTANCE Tuberculosis, caused by Mycobacterium tuberculosis, imposes a major global health burden, surpassing HIV and malaria in annual deaths. The ClpP1P2 proteolytic complex and its cofactor ClpX are attractive drug targets, but their precise cellular functions are unclear. This work confirms ClpX’s essentiality and describes a novel interaction between ClpX and SSB, a component of the DNA replication machinery. Further, we demonstrate that a loss of ClpX is sufficient to interrupt DNA replication, suggesting the ClpX-SSB complex may play a role in DNA replication in mycobacteria.

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