DNA polymerase eta undergoes alternative splicing, protects against UV sensitivity and apoptosis, and suppresses MreII-dependent recombination

Polymerase eta (pot TI) is a low-fidelity DNA polymerase that is the produc t of the gene, POLH, associated with the human XP variant disorder in which there is an extremely high level of solar-induced skin carcinogenesis. The complete human genomic sequence spans about 40 kb containing 10 coding exo ns and a cDNA of 2.14 kb; exon 1 is untranslated and is 6 kb upstream from the first coding exon. Using bacterial artificial chromosomes (BACs), the g ene was mapped to human chromosome band 6p21 and mouse band, 17D. The gene is expressed in most tissues, except for very low or undetectable levels in peripheral lymphocytes, fetal spleen,. and adult muscle; exon II, however, is frequently spliced out in normal cells and in almost half the transcrip ts in the testis and fetal liver. Expression of POLH in a multicopy episoma l vector proved nonviable, suggesting that overexpression is toxic. Express ion from chromosomally integrated linear copies using either an EF1-alpha o r CMV promoter was functional, resulting in cell lines with low or high lev els of pol. il protein, respectively. Point mutations in the center of the gene and in a C-terminal cysteine and deletion of exon II resulted. in inac tivation, but addition of a terminal 3 amino acid C-terminal tag, or an N- or C-terminal green fluorescent protein, had no effect on function. A low l evel of expression of pol eta eliminated hMreII recombination and partially restored UV survival, but did! not prevent UV-induced apoptosis, which req uired higher levels of expression. Polymerase eta is therefore involved in S-phase checkpoint and signal transduction pathways that lead to. arrest In S, apoptosis, and recombination. In normal cells, the predominant mechanis m of replication of UV damage Involves pot eta -dependent bypass, and MreII -dependent recombination that acts is a secondary, backup mechanism when ce lls are severely depleted of pot eta. (C) 2001 Wiley Liss, Inc.