Article

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

Zeitschrift fur Naturforschung C (Impact Factor: 0.57). 01/2014; 69(1-2):81-8. DOI: 10.5560/ZNC.2012-0133
Source: PubMed

ABSTRACT Base excision repair (BER) is a key pathway for maintaining genomic stability. A key enzyme in the BER pathway is DNA polymerase beta (polbeta). It has been shown that more than 11% of breast, bladder, esophageal, colon, and gastric cancer samples studied so far exhibit polbeta mutation. A truncated form of polbeta, polbetadelta (exon 11 deletion), identified in a colon tumour sample, exhibited dominant negative activity. Using this polbetadelta as bait, we screened a HeLa cDNA library for any interacting protein(s) in the yeast two-hybrid (Y2H) system. Polbetadelta was cloned into a pGBKT7 vector (pGBKT7-polbetadelta). pGBKT7-polbetadelta was transformed into the yeast strain AH109. Then the cDNA library was co-transformed into AH109/pGBKT7-polbetadelta 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 polbeta (XRCC1, MGC5306, and AP endonuclease 1). The only member previously not known to interact with polbeta 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 polbeta which 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 polbeta remains to be seen.

1 Follower
 · 
23 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: DNA polymerase beta (pol beta) provides most of the gap-filling synthesis at apurinic/apyrimidine sites of damaged DNA in the base excision repair pathway. A truncated form of the pol beta protein is expressed in colon and breast cancers. However, the role of the pol beta gene in lung cancer is not known. Thus, we investigated a possible occurrence of pol beta variants in primary lung tumors. The entire cDNA of pol beta obtained by RT-PCR amplification was analyzed for nucleotide sequencing in lung tumor and matched normal lung tissue of the same patient. Three types of variants were detected in squamous, non-small, or large cell carcinomas. The most common variant was a deletion of 87 bp from pol beta cDNA at a site corresponding to exon 11. In addition, a variant exhibiting deletions of 87 and 140 bp together with an insertion of 105 bp was identified in three lung tumors. This is the first report of the occurrence of pol beta variants, possibly splicing variants, in lung cancer. A truncated pol beta protein resulting from variant forms of the gene may impact the function of the enzyme and increase susceptibility to carcinogenesis.
    DNA and Cell Biology 08/1999; 18(7):549-54. DOI:10.1089/104454999315097 · 1.99 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: In the base excision repair pathway, wild-type DNA polymerase beta (WT pol beta) provides most of the gap filling synthesis. A truncated pol beta protein (pol beta Delta), expressed in primary colorectal and breast tumors and in a primary culture of renal cell carcinoma, inhibits the gap filling synthesis and DNA binding activities of WT pol beta. However, a purified recombinant pol beta Delta does not inhibit a purified WT pol beta. To determine the dominant inhibitory activity of pol beta Delta, we examined interactions of purified pol beta Delta with X-ray cross complementing group 1 (XRCC1), poly(ADP-ribose) polymerase (PARP), and apurinic endonuclease (Ape) proteins. All of these proteins interact with pol beta Delta in vitro and in vivo. The pol beta Delta protein can fill one nucleotide gap by inserting a base at the AP site, whereas a presumed binary complex of pol beta Delta and XRCC1 cannot. However, this binary complex not only suppresses gap filling synthesis activity of WT pol beta but also binds more strongly to gapped DNA than WT pol beta bound to XRCC1. These results are the first to suggest that XRCC1 is directly involved in the dominant negative activity of truncated pol beta, possibly leading to the genomic instability characteristic of tumor cells.
    Biochemistry 07/2001; 40(30):9005-9013. DOI:10.1021/bi0028789 · 3.19 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In eukaryotic cells, DNA polymerase beta (polbeta) carries out base-excision repair (BER) required for DNA maintenance, replication, recombination, and drug resistance. A specific deletion in one allele in the coding sequence of the polbeta gene occurs in colorectal and breast carcinomas. The 87-bp deleted region encodes amino acid residues 208-236 in the catalytic domain of the enzyme. Here, we report evidence for expression of the wild-type (WT) and the truncated polbeta proteins in colorectal tumors. To elucidate the potential functional consequences of polbeta truncation, stable HeLa cell lines were established from cloned WT and variant polbetaDelta208-236. Cells expressing the variant protein exhibited substantially decreased BER activity. To test our hypothesis that truncated polbeta may disrupt the function of the WT enzyme, we stably transfected mouse embryonic fibroblast 16.3 cells with polbetaDelta208-236 cDNA. Reverse transcription-PCR and Western blot analyses showed that the new cell line, 16.3DeltaP, expresses the WT and the truncated polbeta mRNA and proteins. BER and binding activities were undetectable in these cells. Furthermore, in vivo the 16.3DeltaP cells were more sensitive to N-methyl-N'-nitro-N-nitrosoguanidine than the 16.3 cells. On adding increasing amounts of 16.3DeltaP protein extracts, the BER and DNA binding activities of extracts of the parent 16.3 cell line progressively declined. These results strongly suggest that truncated polbeta acts as a dominant negative mutant. The defective polbeta may facilitate accumulation of mutations, leading to the expression of a mutator phenotype in tumor cells.
    Proceedings of the National Academy of Sciences 10/1997; 94(19):10324-9. DOI:10.1073/pnas.94.19.10324 · 9.81 Impact Factor