Identification of an Endoplasmic Reticulum Membrane Protein Interacting with DNA Polymerase Beta by a Yeast Two-Hybrid Screen

Kakali Panda, Nandan Bhattacharyya, Kalyani Khanra
2014 Zeitschrift für Naturforschung C - A Journal of Biosciences  
Base excision repair (BER) is a key pathway for maintaining genomic stability. A key enzyme in the BER pathway is DNA polymerase beta (polβ). It has been shown that more than 11% of breast, bladder, esophageal, colon, and gastric cancer samples studied so far exhibit polβ mutation. A truncated form of polβ, polβΔ (exon 11 deletion), identified in a colon tumour sample, exhibited dominant negative activity. Using this poβΔ as bait, we screened a HeLa cDNA library for any interacting protein(s)
more » ... the yeast two-hybrid (Y2H) system. PolβΔ was cloned into a pGBKT7 vector (pGBKT7-polβΔ). pGBKT7-polβΔ was transformed into the yeast strain AH109. Then the cDNA library was co-transformed into AH109=pGBKT7-polβΔ and screened by the selection procedure. The yeast-purified plasmids were transformed into Escherichia coli. Plasmid DNA was isolated from the colonies, purified, digested with Sma I and Sal I, and the fragments were sequenced. Four positive clones were obtained. Out of these, three proteins were already known to interact with polβ (XRCC1, MGC5306, and AP endonuclease 1). The only member previously not known to interact with polβ was phosphatidylinositol glycosylase type S (PIGS). PIGS is a 64-kDa membrane protein, encoded in chromosome 17. The PIGS protein interacts also with wild-type polβwhich was confirmed by co-immunoprecipitation and Western blot analysis. The role of the newly identified protein in the dominant negative function of the variant form of polβ remains to be seen.
doi:10.5560/znc.2012-0133 pmid:24772827 fatcat:iigpb3xb4bb5bjhrhw5ugc33x4