First Evidence of DNA Recombinant Activity of ISCR1

PORTO AYELEN; AYALA JUAN; GUTKIND GABRIEL; DI CONZA JOSÉ

San Francisco

Congreso; 52nd Annual Interscience Conference on Antimicrobial Agents and Chemotherapy; 2012

ASM

Background: It has been proposed that ISCR1 (insertion sequence common region) are IS91-like transposable elements. Alignments of the amino acid sequence of the putative recombinases of ISCR1 and IS91 show key amino acid motifs of the transposition proteins. IS91 is an unusual insertion sequence, employing a rolling circle transposition. This transposition system needs an origin of replication (oriIS) and a replication terminator (terIS); ISCR1, as part of complex class 1 integrons, includes oriIS but lacks terIS. Methods: The putative ISCR1-encoded recombinase open reading frame (ORF513) was inserted into a T7expression vector, pET28b+, overproduced in Escherichia coli BL21 (DE3) and purified by immobilized metal ion affinity chromatography (IMAC). Mobility shift experiments were performed using 6FAM and Cy5 labeled DNA fragments. The purified protein and DNA fragments were mixed in an HEPES buffer, and immediately loaded onto a 6% polyacrylamide gel in modified TAE buffer and electrophoresed at 5 mA/gel. Following electrophoresis, fluorophore-marked DNA fragments in the gel were detected using a Typhoon 9410 scan fluorescence imager. Results: After incubating ORF513 protein with different DNA fragments containing the oriIS and terIS sites of some IS91-like elements and flanking regions of oriISCR1, it was detected evidence of DNA-protein interaction between ORF513 and oriISCR1 with upstream and downstream regions. A fact to highlight is that, in comparison, the mobility changes observed on oriISCR1 are stronger than on other oriIS and terIS sequences tested and even more when the oriISCR1 includes its surrounding regions. Conclusions: In this study we used gel shift experiments to evaluate the ability of ORF513 to bind specific DNA sequences containing oriIS and terIS sites and their flanking regions. We obtained data of a DNA-binding activity for a purified extract of ORF513 on oriISCR1.To the best of our knowledge, this is the first experimental description related to the proposed enzymatic activity for this protein.