Equilibrium and kinetic binding analysis of the N-terminal domain of the Pf1 gene 5 protein and its interaction with single-stranded DNA

ABSTRACT SV40 large T antigen (T-ag) is a multifunctional protein that successively binds to 5′-GAGGC-3′ sequences in the viral origin of replication, melts the origin, unwinds DNA ahead of the replication fork, and interacts with host DNA replication factors to promote replication of the simian virus 40 genome. The transition of T-ag from a sequence-specific binding protein to a nonspecific helicase involves its assembly into a double hexamer whose formation is likely dictated by the propensity of T-ag to oligomerize and its relative affinities for the origin as well as for nonspecific double- and single-stranded DNA. In this study, we used a sensitive assay based on fluorescence anisotropy to measure the affinities of wild-type and mutant forms of the T-ag origin-binding domain (OBD), and of a larger fragment containing the N-terminal domain (N260), for different DNA substrates. We report that the N-terminal domain does not contribute to binding affinity but reduces the propensity of the OBD to self-associate. We found that the OBD binds with different affinities to its four sites in the origin and determined a consensus binding site by systematic mutagenesis of the 5′-GAGGC-3′ sequence and of the residue downstream of it, which also contributes to affinity. Interestingly, the OBD also binds to single-stranded DNA with an ∼10-fold higher affinity than to nonspecific duplex DNA and in a mutually exclusive manner. Finally, we provide evidence that the sequence specificity of full-length T-ag is lower than that of the OBD. These results provide a quantitative basis onto which to anchor our understanding of the interaction of T-ag with the origin and its assembly into a double hexamer.

Download Full-text

Mcm4 C-terminal domain of MCM helicase prevents excessive formation of single-stranded DNA at stalled replication forks

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0805307105 ◽

2008 ◽

Vol 105 (35) ◽

pp. 12973-12978 ◽

Cited By ~ 17

Author(s):

N. Nitani ◽

C. Yadani ◽

H. Yabuuchi ◽

H. Masukata ◽

T. Nakagawa

Keyword(s):

Replication Forks ◽

Single Stranded Dna ◽

Terminal Domain ◽

Mcm Helicase ◽

Stalled Replication Forks

Download Full-text

Bacillus thuringiensis Cry4Ba Insecticidal ToxinExploits Leu615 in Its C-terminal Domain to Interact with a Target Receptor—Aedes aegypti Membrane-Bound Alkaline Phosphatase

Toxins ◽

10.3390/toxins13080553 ◽

2021 ◽

Vol 13 (8) ◽

pp. 553

Author(s):

Anon Thammasittirong ◽

Sutticha Na-Ranong Thammasittirong ◽

Chompounoot Imtong ◽

Sathapat Charoenjotivadhanakul ◽

Somsri Sakdee ◽

...

Keyword(s):

Alkaline Phosphatase ◽

Bacillus Thuringiensis ◽

Aedes Aegypti ◽

Receptor Binding ◽

Sandwich Elisa ◽

Full Length ◽

Terminal Domain ◽

Binding Analysis ◽

Membrane Bound ◽

Larval Toxicity

In addition to the receptor-binding domain (DII), the C-terminal domain (DIII) of three-domain Cry insecticidal d-endotoxins from Bacillus thuringiensis has been implicated in target insect specificity, yet its precise mechanistic role remains unclear. Here, the 21kDa high-purity isolated DIII fragment derived from the Cry4Ba mosquito-specific toxin was achieved via optimized preparative FPLC, allowing direct rendering analyses for binding characteristics toward its target receptor—Aedes aegypti membrane-bound alkaline phosphatase (Aa-mALP). Binding analysis via dotblotting revealed that the Cry4Ba-DIII truncate was capable of specific binding to nitrocellulose-bound Aa-mALP, with a binding signal comparable to its 65kDa Cry4Ba-R203Q full-length toxin. Further determination of binding affinity via sandwich ELISA revealed that Cry4Ba-DIII exhibited a rather weak binding to Aa-mALP with a dissociation constant (Kd) of ≈1.1 ×10−7 M as compared with the full-length toxin. Intermolecular docking between the Cry4Ba-R203Q active toxin and Aa-mALP suggested that four Cry4Ba-DIII residues, i.e., Glu522, Asn552, Asn576, and Leu615, are potentially involved in such toxin–receptor interactions. Ala substitutions of each residue (E522A, N552A, N576A and L615A) revealed that only the L615A mutant displayed a drastic decrease in biotoxicity against A. aegypti larvae. Additional binding analysis revealed that the L615A-impaired toxin also exhibited a reduction in binding capability to the surface-immobilized Aa-mALP receptor, while two bio-inactive DII-mutant toxins, Y332A and F364A, which almost entirely lost their biotoxicity, apparently retained a higher degree of binding activity. Altogether, our data disclose a functional importance of the C-terminal domain of Cry4Ba for serving as a potential receptor-binding moiety in which DIII-Leu615 could conceivably be exploited for the binding to Aa-mALP, highlighting its contribution to toxin interactions with such a target receptor in mediating larval toxicity.

Download Full-text

SSB Protein—Its Interaction with DNA and Use as a Tool in Studying Genome Structure

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100098745 ◽

1981 ◽

Vol 39 ◽

pp. 448-451

Author(s):

Susan Chrysegelos ◽

Kathi Dunn ◽

Jack Griffith ◽

Marcia Manning ◽

Claire Moore

Keyword(s):

Genome Structure ◽

Duplex Dna ◽

Single Stranded Dna ◽

Functional Roles ◽

E Coli ◽

Gene 5 Protein ◽

Nucleoprotein Complex ◽

Infected Cells ◽

Gene 32

A protein which binds tightly to single stranded DNA but not duplex DNA was first isolated from Escherichia coli (E. coli) by Sigal et. al and is called SSB for single stranded DNA binding protein. Together with SSB the gene 32 protein of T4 infected E. coli cells, and the gene 5 protein of phage M13 infected cells, are the best characterized members of the helix destabilizing family of proteins. They all share the properties (reviewed by Kbrnberg) of binding very tightly and cooperatively to single stranded DNA, of binding somewhat less well to single stranded RNA, and of binding poorly if at all to duplex DNA or RNA. In binding single stranded polynucleotides these proteins disrupt all secondary structure yielding a linear nucleoprotein complex. The details of binding however are very different from one protein to another and must reflect their functional roles in vivo.Physical studies of SSB have showi it to exist as a 75,000 dalton tetramer in solution which is assumed to be the active unit.

Download Full-text

109 Preferential binding of the fd gene 5 protein to a structured form of the single-stranded DNA sequence d(GT5G4CT4C)

Biochemical Society Transactions ◽

10.1042/bst025s643 ◽

1997 ◽

Vol 25 (4) ◽

pp. S643-S643

Author(s):

Antony W. Oliver ◽

G. Geoff Kneale

Keyword(s):

Dna Sequence ◽

Single Stranded Dna ◽

Gene 5 Protein ◽

Preferential Binding

Download Full-text

Single stranded DNA annealing is a conserved activity of telomere resolvases

PLoS ONE ◽

10.1371/journal.pone.0246212 ◽

2021 ◽

Vol 16 (2) ◽

pp. e0246212

Author(s):

Siobhan L. McGrath ◽

Shu Hui Huang ◽

Kerri Kobryn

Keyword(s):

Dna Binding ◽

Dna Cleavage ◽

Bacterial Species ◽

Dna Binding Protein ◽

Tyrosine Recombinase ◽

Single Stranded Dna ◽

Terminal Domain ◽

Topoisomerase Ib ◽

Replication Intermediates

Bacterial species of the genera Agrobacterium and Borrelia possess chromosomes terminated by hairpin telomeres. Replication produces dimeric replication intermediates fused via replicated telomere junctions. A specialized class of enzymes, referred to as telomere resolvases, promotes the resolution of the replicated intermediate into linear monomers terminated by hairpin telomeres. Telomere resolution is catalyzed via DNA cleavage and rejoining events mechanistically similar to those promoted by topoisomerase-IB and tyrosine recombinase enzymes. Examination of the borrelial telomere resolvase, ResT, revealed unanticipated multifunctionality; aside from its expected telomere resolution activity ResT possessed a singled-stranded DNA (ssDNA) annealing activity that extended to both naked ssDNA and ssDNA complexed with its cognate single-stranded DNA binding protein (SSB). At present, the role this DNA annealing activity plays in vivo remains unknown. We have demonstrated here that single-stranded DNA annealing is also a conserved property of the agrobacterial telomere resolvase, TelA. This activity in TelA similarly extends to both naked ssDNA and ssDNA bound by its cognate SSB. TelA’s annealing activity was shown to stem from the N-terminal domain; removal of this domain abolished annealing without affecting telomere resolution. Further, independent expression of the N-terminal domain of TelA produced a functional annealing protein. We suggest that the apparent conservation of annealing activity in two telomere resolvases, from distantly related bacterial species, implies a role for this activity in hairpin telomere metabolism. Our demonstration of the separation of the telomere resolution and annealing activities of TelA provides a platform for future experiments aimed at identifying the role DNA annealing performs in vivo.

Download Full-text