Studies on enzumes involved in DNA-Protein interactions in hyperthermophilic archaea

Abstract

DNA binding proteins play a significant role in the cellular activities like DNA packaging, replication, transcription, genome expression and DNA repair. The study of DNA binding proteins and their interactions during various processes helps in understanding the principles of binding and provide insights about how bases are recognized, interacted and modified. Since archaea are the residents of extreme conditions, it is more interesting to study such proteins and their interactions with DNA in these organisms to understand the mechanisms involved in maintaining DNA intact at higher temperatures. Two such DNA binding proteins were selected for the present study i.e. flap endonuclease1 and histone proteins. Genome sequences of Thermococcus kodakarensis contain an open reading frame, TK1281, annotated as flap endonuclease1. TK1281 was cloned and expressed in Escherchia coli in soluble and active form. The recombinant protein was purified to homogeneity on SDS-PAGE with the observed mass of 38 kDa by HiTrap Heparin column followed by ion exchange column chromatography. Gel filtration chromatography showed that the protein was monomeric in nature with calculated molecular mass of 38 kDa, similar to the mass observed on SDS-PAGE. TK1281 preferably cleaved double stranded DNA with 5ʹ-flap substrate. The optimum temperature for its cleavage activity was 80 °C and optimum pH was 7.5. The cleavage activity of TK1281 was activated in the presence of divalent metal ions (Co2 , Zn2+ and Mg2+). The cleavage activity of most of the flap endonucleases was supported either by Mg2+ or Mn2+ . In contrast, TK1281 exhibited highest activity in the presence of Co2+ followed by Zn2+ and then Mg2+. Interestingly, no activation was observed in the presence of Mn2+ . The Vmax and Km values were 278 nmol/min/mg and 37 µM, respectively. ii Thermostability assay depicted that protein was stable up to 3 hours at 80 °C. This result was further confirmed by the circular dichroism. The genome sequences of Pyrococcus furiosus and Pyrococcus horikoshii contained two open reading frames, PF1722 and PHS046, respectively, annotated as histones. These histone homologues were uncharacterized therefore selected for the present study. The genes encoding for PF1722 and PHS046 were initially cloned in pET-21a(+) expression vector however the gene expression was very low and purification was not feasible. So the genes were cloned in pET-28a(+) expression vector independently so that gene product can be purified by Nickel-NTA column chromatography. The recombinant histone proteins were produced in soluble form and purified to apparent homogeneity on SDS-PAGE. The apparent molecular masses of these proteins were ̴7 kDa. When the DNA compacting ability of these proteins was observed by electrophoretic mobility shift assay it was found that these proteins were active in compacting DNA. Highest compacting ability was found at protein to DNA ration of 1. When temperature dependency was analyzed, it was observed that PF1722 was able to compact DNA at temperature up to 80 °C. No compacting was observed at 90 °C. While PHS046 was able to compact DNA up to 70 °C and no compacting was observed at higher temperatures. Proteins were highly thermo stable with less compacting observed at low concentration of KCl (200 mM) but at higher concentration (1000 mM) compacting ability was increased as presence of high salt concentration helps in prevention of DNA denaturation. Thus TK1281 is a highly stable nuclease that has specific cleavage activity for 5′-flap from double-stranded DNA in the presence of Co2+. PF1722 and PHS046 are active proteins for compacting DNA at protein to DNA ratio of 0.3 with ability to compact DNA at higher temperatures. The availability of pure proteins in abundant amount will help in iii the further investigation of structural studies that allow understanding the catalytic mechanisms of proteins involved in interaction of proteins with DNA. Future studies will also be helpful in elucidating the properties of proteins for maintaining intact structures of DNA at higher temperatures in archaeal species.