It is likely that restoration of 3-methylcytosine and additional similar alkylation adducts in most cells is mediated by additional members of the Abh family, such as ALKBH2, or by alternate mechanisms that are yet to be identified. dealkylation. In cell lines that are dependent on ALKBH3 and ASCC3 for alkylation damage resistance, loss of ALKBH3 or ASCC3 prospects to improved 3-methylcytosine and reduced cell proliferation, which correlates with pH2A.X and 53BP1 foci formation. Our data provide a molecular mechanism N106 by which ALKBH3 collaborates with ASCC to keep up genomic integrity inside a cell type specific manner. Intro The genome is definitely continually subjected to numerous harmful insults, such as ionizing radiation and nucleic acid modifying compounds. Alkylation is one of the mechanisms by which nucleic acids can be altered and its continuous restoration is important to genomic integrity. Alkylating providers are ubiquitous in the environment, but a major source of alkylation damage is thought to be due to normal cellular rate of metabolism (Ringvoll et al., 2006; Rydberg and Lindahl, 1982; Sedgwick, 2004). In mammalian cells, several possible sources of non-enzymatic methylation by methyl group donors exist, but only the universal methyl donor absence of AlkB prospects to a moderate hypersensitivity to MMS and a significantly increased rate of MMS-mediated base substitution mutations (Delaney and Essigmann, 2004). Mammalian cells have at least eight AlkB family members, ALKBH1 through ALKBH8 (Drablos et al., 2004). Only two of the corresponding human proteins, ALKBH2 and ALKBH3, have been convincingly shown to possess repair activity similar to the AlkB protein (Aas et al., 2003; Duncan et al., 2002; Lee et al., 2005). Biochemical experiments suggest that ALKBH2 prefers double-stranded DNA (dsDNA) substrates, while ALKBH3 prefers single-stranded DNA (ssDNA) substrates and also demethylates 1meA and 3meC in RNA (Aas et al., 2003). Mouse knockout studies have exhibited that the loss of causes accumulation of 1meA in genomic DNA as well as enhanced MMS sensitivity (Ringvoll et al., 2006). N106 In the same study, these phenotypes were not observed in the knockout mice (Ringvoll et al., 2006). These results, as well as the preference of ALKBH3 for single-stranded substrates, have raised the question as to whether ALKBH3 has any role in maintaining genomic integrity (Falnes et al., 2004; Ringvoll et al., 2006), although redundancy amongst AlkB family members remains a possibility. Furthermore, despite considerable characterization of these enzymes, how they access alkylated DNA substrates and whether they play a general or lineage-specific role under physiological or pathological conditions remain unanswered. Here, we investigate the biological function and molecular mechanism of action of ALKBH3, initiated by purification of ALKBH3 and its associated partner proteins from human cells. We found that ALKBH3 associated with an uncharacterized complex, the Activating Transmission Co-integrator Complex (ASCC). Interestingly, ALKBH3 has recently been shown to be overexpressed in a subset of cancers, including prostate and non-small cell lung carcinoma (Konishi et al., 2005; Tasaki et al., 2011). N106 We demonstrate that in tumor cell lines that overexpress ALKBH3, both ALKBH3 and ASCC3, the largest subunit of ASCC, are important for alkylation damage resistance. Importantly, ASCC3 encodes a 3-5 DNA helicase, an activity which is present in the ALKBH3 complex and co-purifies with ASCC3. We show that ASCC3 unwinds DNA to generate the single-stranded substrate needed for ALKBH3-mediated DNA repair, thus exposing a molecular mechanism that involves coupling of a helicase and a dealkylating enzyme in the maintenance of genomic integrity. Strikingly, we found that loss of ALKBH3 or ASCC3 significantly reduces cell proliferation, both in N106 culture and in a xenograft model, of tumor cell lines dependent on this pathway for alkylation damage resistance. This correlated with the accumulation of endogenous 3meC in genomic DNA, as well as formation of pH2A.X and 53BP1 foci, suggesting activation of the DNA damage response. Our findings uncover a DNA repair enzyme complex with a potential role in specific tumor types, and spotlight a molecular mechanism by which a dealkylating enzyme repairs damaged DNA to maintain genomic integrity. Results Biochemical characterization of the ALKBH3 complex To gain greater insight into the function of ALKBH3, we purified the ALKBH3 complex from nuclear extracts of 293T cells using the sequential double-affinity tag purification technique explained previously (Nakatani and Ogryzko, 2003). The producing ALKBH3 complex (Physique 1a) was subjected to tandem mass spectrometry. Besides ALKBH3 itself, we recognized numerous peptides corresponding to three subunits of the Activating Transmission Co-integrator Complex (ASCC; Physique 1a and Supplemental Table 1a). The three subunits, ASCC1, ASCC2, and ASCC3 (also named p50, VEGFA p100, and p300, respectively), were previously found to be associated with a transcriptional activation complex (Jung et al., 2002). However, the precise molecular function of this complex is usually thus far unclear. The largest subunit, ASCC3, is usually predicted to encode a superfamily II-type helicase, although to our knowledge, no enzymatic activity has been yet explained. We also purified the ALKBH3 complex from HeLa cells and found all three components of the ASCC complex (data not shown). Open in a separate window Physique 1 ALKBH3 associates with the mammalian ASCC complex. (a) Silver staining of.