DNA polymerase η undergoes alternative splicing, protects against UV sensitivity and apoptosis, and suppresses Mre11‐dependent recombination

Abstract

Polymerase eta (pol eta) 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 I 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 eta 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 hMre11 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 pol eta-dependent bypass, and Mre11-dependent recombination that acts is a secondary, backup mechanism when cells are severely depleted of pol eta.

p53 suppression overwhelms DNA polymerase η deficiency in determining the cellular UV DNA damage response

“…In previous studies, it was observed that XP-V cells with impaired p53 employed an MRNdependent recombination pathway in response to UV damage [17,25,27,28], implying DSBs are generated during replication arrest [49,[52][53][54]. We observed that p53 suppression leads to a similar increase in the frequency of normal and XP-V cells with Mre11 foci, even without UV irradiation.…”

“…Each of the three means of inactivating p53 are distinct, although all appear to destabilize the S phase in UV damaged cells and contribute to higher levels of DNA breakage (Fig 4) and increased importance of the ATR signal transduction pathway. XP-V cells transformed with SV40 uniquely show increased UV sensitivity when ATR is inhibited by caffeine [7,35,43]; this may be due to the elevated levels of p53 in equilibrium with large T that mimic high levels of damage and induce an apoptotic response [28]. XP-V cells transformed by HPV16(E6/E7) are highly UV sensitive without further sensitization [23], suggesting that there are additional targets for the E6 ubiquitin ligase that play supporting roles in the UV response.…”

“…XP-V cells, in which p53 function was abrogated due to viral transformation by HPV16 (E6) [20,21] or SV40 [22], demonstrated a dramatic increase in sensitivity to the lethal and cytogenetic effects of UV light [23]. These p53-compromised cells employed Mre11 and Rad51-dependent recombination pathways to recover from UV damage [17,[24][25][26][27][28], implying that replication forks arrested by UV damage rapidly undergo a collapse leading to double strand breakage. These cells also show increased sister chromatid exchanges indicative of increased rates of recombination during the S phase [23].…”

Ablation of XP-V gene causes adipose tissue senescence and metabolic abnormalities

“…It is not clear from the available literature whether patients with XP-V are prone to the development of obesity and the metabolic syndrome. Given that variation in the expression level of the XP-V gene affects the sensitivity to DNA-damaging agents in various tissues (10,(38)(39)(40), an epidemiological study will be necessary to examine the correlation between the pol η expression level and the metabolic syndrome. In addition to pol η, other translesion synthesis polymerases have been shown to handle various types of DNA lesions (41), and it will be interesting to determine whether KO mice for other lesion bypass polymerases are prone to obesity and metabolic abnormalities.…”

“…Patients with XP-V are highly sensitive to sunlight and prone to cutaneous cancer (9). In addition to skin, pol η is expressed in most tissues (10). The expression of pol η correlates with the effectiveness of anticancer therapeutic agents that exert their activity by introducing DNA lesions that block the progression of DNA replication (11).…”