title: Sphingosine, a modulator of human translesion DNA polymerase activity.
abstract: 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.
authors.affiliation: From the Departments of Pathology and Biochemistry, The Gottstein Memorial Cancer Research Laboratory, University of Washington, Seattle, Washington 98195.
authors.forename: Ashwini S
authors.initials: AS
authors.lastname: Kamath-Loeb
authors.affiliation: From the Departments of Pathology and Biochemistry, The Gottstein Memorial Cancer Research Laboratory, University of Washington, Seattle, Washington 98195.
authors.forename: Sharath
authors.initials: S
authors.lastname: Balakrishna
authors.affiliation: the Department of Medicinal Chemistry, Mass Spectrometry Center, University of Washington, Seattle, Washington 98195.
authors.forename: Dale
authors.initials: D
authors.lastname: Whittington
authors.affiliation: From the Departments of Pathology and Biochemistry, The Gottstein Memorial Cancer Research Laboratory, University of Washington, Seattle, Washington 98195.
authors.forename: Jiang-Cheng
authors.initials: JC
authors.lastname: Shen
authors.affiliation: Biostatistics and Center for Biomedical Statistics, University of Washington, Seattle, Washington 98195.
authors.forename: Mary J
authors.initials: MJ
authors.lastname: Emond
authors.affiliation: the Open Innovation Center for Drug Discovery, University of Tokyo, Tokyo 113-0033, Japan.
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Okabe
authors.affiliation: the Department of Genome Dynamics, Research Institute of Environmental Medicine, Nagoya University, Nagoya 464-8601, Japan.
authors.forename: Chikahide
authors.initials: C
authors.lastname: Masutani
authors.affiliation: the Institute for Biomolecular Science, Faculty of Science, Gakushuin University, Tokyo 171-8588, Japan, and.
authors.forename: Fumio
authors.initials: F
authors.lastname: Hanaoka
authors.affiliation: the Laboratory Animal Resource Center, University of Tsukuba, Tsukuba 305-8575, Japan.
authors.forename: Susumu
authors.initials: S
authors.lastname: Nishimura
authors.affiliation: From the Departments of Pathology and Biochemistry, The Gottstein Memorial Cancer Research Laboratory, University of Washington, Seattle, Washington 98195, laloeb@uw.edu.
authors.forename: Lawrence A
authors.initials: LA
authors.lastname: Loeb
mesh_terms: D001483:Base Sequence; D004249:DNA Damage; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008081:Liposomes; D013110:Sphingosine / Q000502:physiology*; D000099270:Y-Family DNA Polymerases
chemical_list: D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase; D008081:Liposomes; D013110:Sphingosine; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases
keywords: DNA Damage; DNA Polymerase; Drug Screening; Genomic Instability; Sphingolipid; Sphingosine; Translesion