Translesion (TLS)
DNA polymerases are specialized, error-prone
enzymes that synthesize
DNA across bulky, replication-stalling
DNA adducts. In so doing, they facilitate the progression of
DNA synthesis and promote cell proliferation. To potentiate the effect of
cancer chemotherapeutic regimens, we sought to identify inhibitors of TLS
DNA polymerases. We screened five libraries of ∼ 3000 small molecules, including one comprising ∼ 600
nucleoside analogs, for their effect on primer extension activity of
DNA polymerase η (Pol η). We serendipitously identified
sphingosine, a
lipid-signaling molecule that robustly stimulates the activity of Pol η by ∼ 100-fold at low micromolar concentrations but inhibits it at higher concentrations. This effect is specific to the Y-family
DNA polymerases, Pols η, κ, and ι. The addition of a single
phosphate group on
sphingosine completely abrogates this effect. Likewise, the inclusion of other
sphingolipids, including
ceramide and
sphingomyelin to extension reactions does not elicit this response.
Sphingosine increases the rate of correct and incorrect
nucleotide incorporation while having no effect on polymerase processivity. Endogenous Pol η activity is modulated similarly as the recombinant
enzyme. Importantly,
sphingosine-treated cells exhibit increased lesion bypass activity, and
sphingosine tethered to
membrane lipids mimics the effects of free
sphingosine. Our studies have uncovered
sphingosine as a modulator of TLS
DNA polymerase activity; this property of
sphingosine may be associated with its known role as a signaling molecule in regulating cell proliferation in response to cellular stress.