Abstract
Helicases are ubiquitous enzymes that can transiently catalyze the unwinding of nucleic acid duplexes using NTP hydrolysis as the source of energy. They play vital roles in nearly all DNA metabolic processes, including DNA replication, recombination and repair. The defects in some specific DNA helicases can cause genetic diseases, and such helicases have been shown to play roles in protecting cells from DNA damaging agents.
The Pif1 subfamily is a 5' to 3' DNA helicase, which belongs to SF1 superfamily, is found to be conserved from yeast to mammals, suggesting important function for genomic maintenance. In order to elucidate the function of human Pif1 helicase, we cloned hPif1 helicase from HeLa cDNA library. The PIF1 cDNA encoded a 69 kDa protein consisting of 641 amino acid residues. The PIF1 gene consisted of 13 exons located on chromosome 15q22. RT-PCR using commercially available muti-tissue cDNA panels revealed that hPIF1 was ubiquitously expressed in all the tested 16 tissues, higher in the thymus, spleen, testis, and very low in the pancreas, prostate, peripheral leukocytes. For further analysis of the gene product, we purified the full-length recombinant proteins from over produced E. coli cells. We demonstrated that the recombinant protein has a DNA-dependent ATPase activity, and also has 5' to 3' helicase activity, which is much stimulated by fork-structured substrates.
