DNA polymerase η 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 product 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 exons 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 gene 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 transcripts in the testis and fetal liver. Expression of POLH in a multicopy episomal vector proved nonviable, suggesting that overexpression is toxic. Expression from chromosomally integrated linear copies using either an EF1-alpha or CMV promoter was functional, resulting in cell lines with low or high levels 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 inactivation, 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 level of expression of pol eta eliminated hMreII recombination and partially restored UV survival, but did! not prevent UV-induced apoptosis, which required 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 mechanism of replication of UV damage Involves pot eta -dependent bypass, and MreII-dependent recombination that acts is a secondary, backup mechanism when cells are severely depleted of pot eta. (C) 2001 Wiley Liss, Inc.