4/2/2023 0 Comments Serial cloner xdna pet28aThese 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. In the same study, these phenotypes were not observed in the AlkBh3 knockout mice ( Ringvoll et al., 2006). Mouse knockout studies have demonstrated that the loss of AlkBh2 causes accumulation of 1meA in genomic DNA as well as enhanced MMS sensitivity ( Ringvoll et al., 2006). 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). coli AlkB protein in vitro ( Aas et al., 2003 Duncan et al., 2002 Lee et al., 2005). Only two of the corresponding human proteins, ALKBH2 and ALKBH3, have been convincingly shown to possess repair activity similar to the E. Mammalian cells have at least eight AlkB family members, ALKBH1 through ALKBH8 ( Drablos et al., 2004). coli, absence of AlkB leads to a moderate hypersensitivity to MMS and a significantly increased rate of MMS-mediated base substitution mutations ( Delaney and Essigmann, 2004). This creates an unstable methyl-iminium intermediate that is spontaneously hydrolyzed to form formaldehyde and a non-alkylated base ( Falnes et al., 2002 Trewick et al., 2002). Bacterial AlkB demethylates alkylated bases such as 1-methyladenine (1meA) and 3-methylcytosine (3meC) by oxidation of the N-linked methyl moiety. coli, AlkB belongs to a large family of non-heme Fe(II) and 2-oxoglutarate-dependent dioxygenases, which catalyze a host of important biological reactions, such as proline hydroxylation and histone demethylation ( Mosammaparast and Shi, 2010 Sundheim et al., 2008). In fact, the estimated endogenous DNA methylation corresponds to an exposure of cells to 20 nM of methyl methanesulfonate (MMS), a potent alkylating agent ( Rydberg and Lindahl, 1982).Ī number of mechanisms exist to repair alkylation damage, including base excision repair (BER), direct reversal by methylguanine methyltransferase (MGMT), and dealkylation repair via the AlkB family of enzymes ( Sedgwick, 2004). In mammalian cells, several possible sources of non-enzymatic methylation by methyl group donors exist, but only the universal methyl donor S-adenosylmethionine has been shown to non-enzymatically methylate DNA ( Barrows and Magee, 1982). Alkylating agents are ubiquitous in the environment, but a major source of alkylation damage is thought to be due to normal cellular metabolism ( Ringvoll et al., 2006 Rydberg and Lindahl, 1982 Sedgwick, 2004). Alkylation is one of the mechanisms by which nucleic acids can be altered and its continuous repair is important to genomic integrity. The genome is continuously subjected to various harmful insults, such as ionizing radiation and nucleic acid modifying compounds. Our data provide a molecular mechanism by which ALKBH3 collaborates with ASCC to maintain genomic integrity in a cell type specific manner. In cell lines that are dependent on ALKBH3 and ASCC3 for alkylation damage resistance, loss of ALKBH3 or ASCC3 leads to increased 3-methylcytosine and reduced cell proliferation, which correlates with pH2A.X and 53BP1 foci formation. ASCC3, the largest subunit of ASCC, encodes a 3′-5′ DNA helicase, whose activity is crucial for the generation of single-stranded DNA upon which ALKBH3 preferentially functions for dealkylation. ALKBH3 is overexpressed in various cancers, and both ALKBH3 and ASCC are important for alkylation damage resistance in these tumor cell lines. We report the purification of the ALKBH3 complex and demonstrate its association with the Activating Signal Co-integrator Complex (ASCC). However, little is known about their functions in mammalian cells. Demethylation by the AlkB dioxygenases represents an important mechanism for repair of N-alkylated nucleotides.
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