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This suggests that, despite their similarities, pol and pol have nonredundant in vivo functions.All known DNA polymerases are believed to share a remarkably high functional and structural similarity. However, each of these enzymes possesses unique features that are crucial to cope with the different DNA transactions encountered while conducting DNA synthesis in vivo.Thus, in addition to the extensive DNA polymerization performed by replicative DNA polymerases, a large and growing number of enzymes have been found to be specialized in largely different and sometimes surprising types of synthesis. Since its discovery, pol has been suggested to play a role in DNA synthesis associated with DNA repair processes in the nucleus of mammalian cells.Indeed, it is accepted today that its polymerization features, combined with its dRP lyase activity, make pol a crucial protein for base excision repair are small, monomeric enzymes that can be found in all realms of life, including archaea, eubacteria, eukaryotes, and viruses. In addition to terminal deoxynucleotidyl transferase and mammalian pol, a large number of family X enzymes have been now characterized in different organisms, including yeast pol IV. However, unlike pol, pol has a BRCT domain at its N terminus, which likely takes part in proteinprotein or proteinDNA interactions. Northern blot analysis reflects that murine and human pol mRNA is highly abundant in testis. Moreover, predominant expression of murine pol in pachytene spermatocytes has led to the hypothesis that it plays a role in DNA repair synthesis coupled to meiotic recombination. Besides having an intrinsic DNA polymerase activity, and the recent demonstration of an intrinsic dRP lyase activity, little is known about the biochemical features of pol. This is mainly because previous studies were limited by the lack of purified protein.Here, we describe the purification of human pol and its basic in vitro polymerization features and discuss novel insights into its cellular function.Human pol cDNA was obtained from human placenta by PCR amplification.A second fragment was obtained by semispecific PCR, using a sense primer derived from the murine sequence close to the initiation codon and an antisense primer derived from the first PCR fragment, which contained the coding sequence corresponding to positions of human pol cDNA.The cDNA sequence was completed by rapid amplification of cDNA ends, a bp long fragment that contained the untranslated region, and the initial portion of the coding region.Therefore, the complete cDNA of human pol contains a total of bp, with a untranslated region of bp, and a untranslated region of bp. Subsequently, the cultured cells were harvested, and the pelleted cells were weighted. Cell debris and alumina were discarded after a min centrifugation at, g.Insoluble material was pelleted by a min centrifugation at, g.polyethyleneimine and sedimented by centrifugation for min at, g.After several washing steps with native binding buffer <a href="http://inhibit09.online/archives/221"></a>   containing increasing concentration of imidazole, pol was eluted at mM imidazole.Polymerase containing fractions were collected, fold diluted with buffer A, bound to a third PC column human pol. mgml BSA, was stored at C.A commercial panel of human tissue cDNA libraries was used as prenormalized cDNA templates.

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