Determination of oligomeric status of HpDprA and its domains.

<p>Elution chromatograms of purified HpDprA at 2 mg/ml and 1 mg/ml <b>(A)</b> concentrations are depicted. Similarly, the size exclusion profiles of HpRF at 2 mg/ml at 1 mg/ml <b>(B)</b> and HpDML1 at 8 mg/ml at 2 mg/ml <b>(C)</b> are depicted. <b>(D)</b> Table shows elution volume (V_e) and corresponding molecular weight of the elution peaks shown in (<b>A, B</b> and <b>C</b>). Elution was monitored using UV absorbance at 230 nm wavelength. The column was equilibrated in the same buffer as the protein samples (1X PBS). Total 500 µl sample was injected for each chromatophic experiment. The apparent molecular mass was calculated from elution volume using molecular weight standards as described in materials and methods.</p

Surface plasmon resonance analysis of HpDprA mutants interaction with DNA.

<p>Different concentrations of <b>(A)</b>HpDprA^R48A/R49Aand <b>(C)</b>HpDprA^{R48A/R49A/K133A} were injected on the single stranded DNA surface in standard buffer containing 150 mM NaCl. Both sample injection and buffer injection were carried out as described in materials and methods. Representative sensorgrams illustrating changes in the response units (the y—axis) as a function of time (the x-axis) are shown. Similarly, binding sensorgrams were obtained for the interaction of dsDNA with <b>(B)</b>HpDprA^R48A/R49Aand <b>(D)</b>HpDprA^{R48A/R49A/K133A}. The concentrations of soluble analytes and affinity constant (K_d) values are indicated in the inset to the figures. The K_d values are determined as described in materials and methods.</p

Sequences of the oligonucleotides used in this study.

<p>Sequences of the oligonucleotides used in this study.</p

Surface plasmon resonance analysis of HpDprA, HpRF and HpDML1 interaction with DNA.

<p>Different concentrations of full length HpDprA <b>(A)</b>HpRF<b>(C)</b> and HpDML1<b>(E)</b> were injected on to the immobilized single stranded DNA (90 mer, ~1000 RU) surface in buffer containing 150 mM NaCl. Both sample injection and buffer injection were carried out as described in materials and methods. Similarly, binding sensorgrams were obtained for the interaction of dsDNA (90 bp, ~1000 RU) with full length HpDprA <b>(B)</b>HpRF<b>(D)</b> and HpDML1<b>(F)</b>. Sensorgrams depicting changes in response unit (the y—axis) as a function of time (the x—axis) are shown. The concentrations of soluble analytes and affinity constant (K_d) values are indicated in the inset to the figures.</p

Secondary structure composition of full length HpDprA and its individual domains.

<p>The secondary structure of individual proteins have been calculated using K2D2, a web server to estimate the α helix and β strand content of a protein from its circular dichroism spectrum and compared with the % α –helix and % β sheet calculated from HpDprA structure published by Wang et. al., (2014). RMSD values depicting goodness of fit has been shown in bracket<b>.</b></p><p>Secondary structure composition of full length HpDprA and its individual domains.</p

Nuclease protection assay.

<p><b>(A)</b> Schematic illustration of nuclease protection assay. ³²P-labeled dsDNA (0.5 nM) either alone (lane 2) or pre-bound with increasing concentrations of HpDprA <b>(B)</b> or HpRF<b>(C)</b> {100, 200, 400, 600, 800, 1000 (nM), lanes 3–8} was incubated for 30 min with 1 unit of DNaseI. Similarly, ³²P-labeled ssDNA (0.5 nM) either alone (lane 2) or pre-bound with above mentioned concentrations of HpDprA <b>(D)</b> or HpRF<b>(E)</b>was incubated for 30 min with 1 unit of mung bean endonuclease. Lane 1: DNAalone.</p