scholarly journals Bacillus subtilis single-stranded DNA-binding protein SsbA is phosphorylated at threonine 38 by the serine/threonine kinase YabT

2017 ◽  
Vol 118 (4) ◽  
Author(s):  
Abderahmane Derouiche ◽  
Dina Petranovic ◽  
Boris Macek ◽  
Ivan Mijakovic
Keyword(s):  
Bacillus Subtilis ◽  
Dna Binding ◽  
Tyrosine Phosphorylation ◽  
Binding Proteins ◽  
Phosphorylation Site ◽  
Dna Binding Proteins ◽  
Mass Spectrometry Analysis ◽  
Single Stranded Dna ◽  
Arginine Residues

Background and purpose: Single-stranded DNA-binding proteins participate in all stages of DNA metabolism that involve single-stranded DNA, from replication, recombination, repair of DNA damage, to natural competence in species such as Bacillus subtilis. B. subtilis single-stranded DNA-binding proteins have previously been found to be phosphorylated on tyrosine and arginine residues. While tyrosine phosphorylation was shown to enhance the DNA-binding properties of SsbA, arginine phosphorylation was not functionally characterized.Materials and methods: We used mass spectrometry analysis to detect phosphorylation of SsbA purified from B. subtilis cells. The detected phosphorylation site was assessed for its influence on DNA-binding in vitro, using electrophoretic mobility shift assays. The ability of B. subtilis serine/threonine kinases to phosphorylate SsbA was assessed using in vitro phosphorylation assays.Results: In addition to the known tyrosine phosphorylation of SsbA on tyrosine 82, we identified a new phosphorylation site: threonine 38. The in vitro assays demonstrated that SsbA is preferentially phosphorylated by the B. subtilis Hanks-type kinase YabT, and phosphorylation of threonine 38 leads to enhanced cooperative binding to DNA.Conclusions: Our findings contribute to the emerging picture that bacterial proteins, exemplified here by SsbA, undergo phosphorylation at multiple residues. This results in a complex regulation of cellular functions, and suggests that the complexity of the bacterial cellular regulation may be underestimated.

scholarly journals Reovirus Protein ςNS Binds in Multiple Copies to Single-Stranded RNA and Shares Properties with Single-Stranded DNA Binding Proteins

2000 ◽  
Vol 74 (13) ◽  
pp. 5939-5948 ◽  
Author(s):  
Anne Lynn Gillian ◽  
Stephen C. Schmechel ◽  
Jonathan Livny ◽  
Leslie A. Schiff ◽  
Max L. Nibert
Keyword(s):  
Dna Binding ◽  
Nucleic Acids ◽  
Binding Proteins ◽  
Atp Hydrolysis ◽  
Nonstructural Protein ◽  
Dna Binding Proteins ◽  
Mobility Shift ◽  
Single Stranded Dna ◽  
Gel Mobility Shift

ABSTRACT Reovirus nonstructural protein ςNS interacts with reovirus plus-strand RNAs in infected cells, but little is known about the nature of those interactions or their roles in viral replication. In this study, a recombinant form of ςNS was analyzed for in vitro binding to nucleic acids using gel mobility shift assays. Multiple units of ςNS bound to single-stranded RNA molecules with positive cooperativity and with each unit covering about 25 nucleotides at saturation. The ςNS protein did not bind preferentially to reovirus RNA over nonreovirus RNA in competition experiments but did bind preferentially to single-stranded over double-stranded nucleic acids and with a slight preference for RNA over DNA. In addition, ςNS bound to single-stranded RNA to which a 19-base DNA oligonucleotide was hybridized at either end or near the middle. When present in saturative amounts, ςNS displaced this oligonucleotide from the partial duplex. The strand displacement activity did not require ATP hydrolysis and was inhibited by MgCl2, distinguishing it from a classical ATP-dependent helicase. These properties of ςNS are similar to those of single-stranded DNA binding proteins that are known to participate in genomic DNA replication, suggesting a related role for ςNS in replication of the reovirus RNA genome.

scholarly journals Complementary roles of Pif1 helicase and single stranded DNA binding proteins in stimulating DNA replication through G-quadruplexes

2019 ◽  
Author(s):  
Melanie A Sparks ◽  
Saurabh P Singh ◽  
Peter M Burgers ◽  
Roberto Galletto
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Binding Proteins ◽  
Protective Role ◽  
Dna Binding Proteins ◽  
Single Stranded Dna ◽  
Fork Stalling ◽  
Pif1 Helicase

Abstract G-quadruplexes (G4s) are stable secondary structures that can lead to the stalling of replication forks and cause genomic instability. Pif1 is a 5′ to 3′ helicase, localized to both the mitochondria and nucleus that can unwind G4s in vitro and prevent fork stalling at G4 forming sequences in vivo. Using in vitro primer extension assays, we show that both G4s and stable hairpins form barriers to nuclear and mitochondrial DNA polymerases δ and γ, respectively. However, while single-stranded DNA binding proteins (SSBs) readily promote replication through hairpins, SSBs are only effective in promoting replication through weak G4s. Using a series of G4s with increasing stabilities, we reveal a threshold above which G4 through-replication is inhibited even with SSBs present, and Pif1 helicase is required. Because Pif1 moves along the template strand with a 5′-3′-directionality, head-on collisions between Pif1 and polymerase δ or γ result in the stimulation of their 3′-exonuclease activity. Both nuclear RPA and mitochondrial SSB play a protective role during DNA replication by preventing excessive DNA degradation caused by the helicase-polymerase conflict.

scholarly journals The Rep Protein of Adeno-Associated Virus Type 2 Interacts with Single-Stranded DNA-Binding Proteins That Enhance Viral Replication

2004 ◽  
Vol 78 (1) ◽  
pp. 441-453 ◽  
Author(s):  
Travis H. Stracker ◽  
Geoffrey D. Cassell ◽  
Peter Ward ◽  
Yueh-Ming Loo ◽  
Bas van Breukelen ◽  
...  
Keyword(s):  
Dna Binding ◽  
Virus Type ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Single Stranded Dna ◽  
Adeno Associated Virus ◽  
Vitro Assay ◽  
Hsv 1

ABSTRACT Adeno-associated virus (AAV) type 2 is a human parvovirus whose replication is dependent upon cellular proteins as well as functions supplied by helper viruses. The minimal herpes simplex virus type 1 (HSV-1) proteins that support AAV replication in cell culture are the helicase-primase complex of UL5, UL8, and UL52, together with the UL29 gene product ICP8. We show that AAV and HSV-1 replication proteins colocalize at discrete intranuclear sites. Transfections with mutant genes demonstrate that enzymatic functions of the helicase-primase are not essential. The ICP8 protein alone enhances AAV replication in an in vitro assay. We also show localization of the cellular replication protein A (RPA) at AAV centers under a variety of conditions that support replication. In vitro assays demonstrate that the AAV Rep68 and Rep78 proteins interact with the single-stranded DNA-binding proteins (ssDBPs) of Ad (Ad-DBP), HSV-1 (ICP8), and the cell (RPA) and that these proteins enhance binding and nicking of Rep proteins at the origin. These results highlight the importance of intranuclear localization and suggest that Rep interaction with multiple ssDBPs allows AAV to replicate under a diverse set of conditions.

Mitochondrial Single-Stranded DNA-Binding Proteins: in Search for New Functions

2001 ◽  
Vol 382 (2) ◽  
Author(s):  
L'ubomír Tomáka ◽  
Jozef Nosek ◽  
Blanka Kucejová
Keyword(s):  
Dna Binding ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Single Stranded Dna

Identification of single-stranded-DNA-binding proteins that interact with muscle gene elements

1991 ◽  
Vol 11 (4) ◽  
pp. 1944-1953
Author(s):  
I M Santoro ◽  
T M Yi ◽  
K Walsh
Keyword(s):  
Dna Binding ◽  
Binding Proteins ◽  
Regulatory Element ◽  
Dna Binding Proteins ◽  
Binding Activity ◽  
Sequence Motif ◽  
Mobility Shift ◽  
Specific Sequence ◽  
Single Stranded Dna ◽  
Muscle Gene

A sequence-specific DNA-binding protein from skeletal-muscle extracts that binds to probes of three muscle gene DNA elements is identified. This protein, referred to as muscle factor 3, forms the predominant nucleoprotein complex with the MCAT gene sequence motif in an electrophoretic mobility shift assay. This protein also binds to the skeletal actin muscle regulatory element, which contains the conserved CArG motif, and to a creatine kinase enhancer probe, which contains the E-box motif, a MyoD-binding site. Muscle factor 3 has a potent sequence-specific, single-stranded-DNA-binding activity. The specificity of this interaction was demonstrated by sequence-specific competition and by mutations that diminished or eliminated detectable complex formation. MyoD, a myogenic determination factor that is distinct from muscle factor 3, also bound to single-stranded-DNA probes in a sequence-specific manner, but other transcription factors did not. Multiple copies of the MCAT motif activated the expression of a heterologous promoter, and a mutation that eliminated expression was correlated with diminished factor binding. Muscle factor 3 and MyoD may be members of a class of DNA-binding proteins that modulate gene expression by their abilities to recognize DNA with unusual secondary structure in addition to specific sequence.

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