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1. Biolayer interferometry for DNA-protein interactions

   https://doi.org/10.1371/journal.pone.0263322  

   Barrows, John K.; Van Dyke, Michael W. (February 2022, PLOS ONE)

   D’Auria, Sabato (Ed.)

   Biolayer interferometry (BLI) is a widely utilized technique for determining macromolecular interaction dynamics in real time. Using changes in the interference pattern of white light reflected off a biosensor tip, BLI can determine binding parameters for protein-protein ( e . g ., antibody-substrate kinetics) or protein-small molecule ( e . g ., drug discovery) interactions. However, a less-appreciated application for BLI analysis is DNA-protein interactions. DNA-binding proteins play an immense role in cellular biology, controlling critical processes including transcription, DNA replication, and DNA repair. Understanding how proteins interact with DNA often provides important insight into their biological function, and novel technologies to assay DNA-protein interactions are of broad interest. Currently, a detailed protocol utilizing BLI for DNA-protein interactions is lacking. In the following protocol, we describe the use of BLI and biotinylated-DNA probes to determine the binding kinetics of a transcription factor to a specific DNA sequence. The experimental steps include the generation of biotinylated-DNA probes, the execution of the BLI experiment, and data analysis by scientific graphing and statistical software ( e . g ., GraphPad Prism). Although the example experiment used throughout this protocol involves a prokaryotic transcription factor, this technique can be easily translated to any DNA-binding protein. Pitfalls and potential solutions for investigating DNA-binding proteins by BLI are also presented. 

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2. The Effects of Packaged, but Misguided, Single-Stranded DNA Genomes Are Transmitted to the Outer Surface of the ϕX174 Capsid

   https://doi.org/10.1128/JVI.00883-21  

   Ogunbunmi, Elizabeth T.; Roznowski, Aaron P.; Fane, Bentley A. (August 2021, Journal of Virology)

   Sandri-Goldin, Rozanne M. (Ed.)

   ABSTRACT Most icosahedral viruses condense their genomes into volumetrically constrained capsids. However, concurrent genome biosynthesis and packaging are specific to single-stranded DNA (ssDNA) viruses. ssDNA genome packaging combines elements found in both double-stranded DNA (dsDNA) and ssRNA systems. Similar to dsDNA viruses, the genome is packaged into a preformed capsid. Like ssRNA viruses, there are numerous capsid-genome associations. In ssDNA microviruses, the DNA-binding protein J guides the genome between 60 icosahedrally ordered DNA binding pockets. It also partially neutralizes the DNA’s negative phosphate backbone. ϕX174-related microviruses, such as G4 and α3, have J proteins that differ in length and charge organization. This suggests that interchanging J proteins could alter the path used to guide DNA in the capsid. Previously, a ϕXG4J chimera, in which the ϕX174 J gene was replaced with the G4 gene, was characterized. It displayed lethal packaging defects, which resulted in procapsids being removed from productive assembly. Here, we report the characterization of another inviable chimera, ϕXα3J. Unlike ϕXG4J, ϕXα3J efficiently packaged DNA but produced noninfectious particles. These particles displayed a reduced ability to attach to host cells, suggesting that internal DNA organization could distort the capsid’s outer surface. Mutations that restored viability altered J-coat protein contact sites. These results provide evidence that the organization of ssDNA can affect both packaging and postpackaging phenomena. IMPORTANCE ssDNA viruses utilize icosahedrally ordered protein-nucleic acids interactions to guide and organize their genomes into preformed shells. As previously demonstrated, chaotic genome-capsid associations can inhibit ϕX174 packaging by destabilizing packaging complexes. However, the consequences of poorly organized genomes may extend beyond the packaging reaction. As demonstrated herein, it can lead to uninfectious packaged particles. Thus, ssDNA genomes should be considered an integral and structural virion component, affecting the properties of the entire particle, which includes the capsid’s outer surface. 

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3. Human Rev1 relies on insert-2 to promote selective binding and accurate replication of stabilized G-quadruplex motifs

   https://doi.org/10.1093/nar/gkab041  

   Ketkar, Amit; Smith, Lane; Johnson, Callie; Richey, Alyssa; Berry, Makayla; Hartman, Jessica H; Maddukuri, Leena; Reed, Megan R; Gunderson, Julie E; Leung, Justin W; et al (February 2021, Nucleic Acids Research)

   null (Ed.)

   Abstract We previously reported that human Rev1 (hRev1) bound to a parallel-stranded G-quadruplex (G4) from the c-MYC promoter with high affinity. We have extended those results to include other G4 motifs, finding that hRev1 exhibited stronger affinity for parallel-stranded G4 than either anti-parallel or hybrid folds. Amino acids in the αE helix of insert-2 were identified as being important for G4 binding. Mutating E466 and Y470 to alanine selectively perturbed G4 binding affinity. The E466K mutant restored wild-type G4 binding properties. Using a forward mutagenesis assay, we discovered that loss of hRev1 increased G4 mutation frequency >200-fold compared to the control sequence. Base substitutions and deletions occurred around and within the G4 motif. Pyridostatin (PDS) exacerbated this effect, as the mutation frequency increased >700-fold over control and deletions upstream of the G4 site more than doubled. Mutagenic replication of G4 DNA (±PDS) was partially rescued by wild-type and E466K hRev1. The E466A or Y470A mutants failed to suppress the PDS-induced increase in G4 mutation frequency. These findings have implications for the role of insert-2, a motif conserved in vertebrates but not yeast or plants, in Rev1-mediated suppression of mutagenesis during G4 replication. 

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4. Conservation of the insert-2 motif confers Rev1 from different species with an ability to disrupt G-quadruplexes and stimulate translesion DNA synthesis

   https://doi.org/10.1039/D3CB00027C  

   Ketkar, Amit; Sewilam, Reham S.; McCrury, Mason J.; Hall, Jaycelyn S.; Bell, Ashtyn; Paxton, Bethany C.; Tripathi, Shreyam; Gunderson, Julie E.C.; Eoff, Robert L. (May 2023, RSC Chemical Biology)

   In some organisms, the replication of G-quadruplex (G4) structures is supported by the Rev1 DNA polymerase. We previously showed that residues in the insert-2 motif of human Rev1 (hRev1) increased the affinity of the enzyme for G4 DNA and mediated suppression of mutagenic replication near G4 motifs. We have now investigated the conservation of G4-selective properties in Rev1 from other species. We compared Rev1 from Danio rerio (zRev1), Saccharomyces cerevisiae (yRev1), and Leishmania donovani (lRev1) with hRev1, including an insert-2 mutant form of hRev1 (E466A/Y470A or EY). We found that zRev1 retained all of the G4-selective prowess of the human enzyme, but there was a marked attenuation of G4 binding affinity for the EY hRev1 mutant and the two Rev1 proteins lacking insert-2 (yRev1 and lRev1). Perhaps most strikingly, we found that insert-2 was important for disruption of the G4 structure and optimal stimulation of processive DNA synthesis across the guanine-rich motif by DNA polymerase kappa (pol κ). Our findings have implications for how Rev1 might contribute to G4 replication in different species spanning the evolutionary tree – signaling the importance of selection for enzymes with robust G4-selective properties in organisms where these non-B DNA structures may fulfill taxa-specific physiological functions. 

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5. 8-Oxoguanine Disrupts G-Quadruplex DNA Stability and Modulates FANCJ AKKQ Peptide Binding

   https://doi.org/10.3390/molecules30163424  

   Campbell, Laura; Lowran, Kaitlin; Cismas, Emma; Wu, Colin G (August 2025, Molecules)

   Guanine-rich nucleic acid sequences can adopt G-quadruplex (G4) structures, which pose barriers to DNA replication and repair. The FANCJ helicase contributes to genome stability by resolving these structures, a function linked to its G4-binding site that features an AKKQ amino acid motif. This site is thought to recognize oxidatively damaged G4, specifically those containing 8-oxoguanine (8oxoG) modifications. We hypothesize that FANCJ AKKQ recognition of 8oxoG-modified G4s (8oxoG4s) depends on the sequence context, the position of the lesion within the G4, and overall structural stability. Using fluorescence spectroscopy, we measured the binding affinities of a FANCJ AKKQ peptide for G4s formed by (GGGT)4, (GGGTT)4, and (TTAGGG)4 sequences. G4 conformation and thermal stability were assessed by circular dichroism spectroscopy. Each sequence was modified to include a single 8oxoG at the first (8oxo1), third (8oxo3), or fifth (8oxo5) guanine position. In potassium chloride (KCl), the most destabilized structures were (GGGT)4 8oxo1, (GGGTT)4 8oxo1, and (TTAGGG)4 8oxo5. In sodium chloride (NaCl), the most destabilized were (GGGT)4 8oxo1, (GGGTT)4 8oxo5, and (TTAGGG)4 8oxo5. FANCJ AKKQ binding affinities varied according to damage position and sequence context, with notable differences for (GGGT)4 in KCl and (TTAGGG)4 in NaCl. These findings support a model in which FANCJ binding to G4 and 8oxoG4 structures is modulated by both the oxidative damage position and the G4 local sequence environment. 

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