Interstrand crosslinks generated from the repair of etheno DNA adducts - Biomolecular crosslinks (CLs) cause critical defects in vital biological processes, such as DNA replication and transcription. DNA–DNA interstrand crosslinks (ICLs) are considered to be the most harmful type of crosslink. Recently, we reported a novel type of ICL produced as a side product during the attempted repair of 1,N6- ethenoadenine (εA) by the human Fe(II)/α-ketoglutarate (α-KG)-dependent enzyme ALKBH2. The ICL is proposed to be generated through covalent bond formation between the epoxide intermediate of εA repair and the exocyclic N6 of adenine or the N4 of cytosine in the complementary strand. The crosslink generated from attempted εA repair was detected in cells by highly sensitive LC-MS techniques, giving biological relevance to the crosslink adducts. Similar to εA, four other etheno adducts have been identified: 3,N4-ethenocytosine, 3,N4- etheno-5-methylcytosine, 1,N2-ethenoguanine, and N2,3-ethenoguanine. These adducts are formed either exogenously from the human carcinogen vinyl chloride and its derivatives or endogenously from products of lipid peroxidation. Several DNA repair pathways for etheno adducts have been reported, including base excision repair (BER) and direct reversal repair (DRR). DRR of etheno lesions is achieved by the AlkB family enzymes, which are α-KG/Fe(II)-dependent. Nine human homologs (designated as ALKBH1-8 and FTO) have been identified to the AlkB protein of E. coli. We will study all five etheno adducts (etheno adductome) for crosslinks formation with four AlkB enzymes (ALKBH2/3/5/FTO). The central hypothesis of this proposal is that four out of the five etheno adducts, but not N2,3-ethenoguanine, form ICLs during the oxidative repair by the ALKBH2/3/5/FTO; these CLs exist in cells, especially in those exposed to vinyl chloride and its derivatives or under inflammation conditions. This type of ICL has a complicated chemical structure with a low abundance in cells. However, similar to other types of ICLs, detrimental biological effects occur even when an ICL presents in minute quantities. To study the complex structure and cellular existence of the ICL, multidisciplinary expertise is required that is usually not possessed by a single lab. Thus, we assembled a strong team including three individual labs with complementary skillsets. In Aim 1 (Li lab), we will study the ICL biochemically by incubating DNA with an AlkB family protein and cofactors and identify the existence of ICLs with LC-MS and gel electrophoresis. We will systematically study the formation of ICL under different sequence contexts. In Aim 2 (Cisneros lab), we will use theoretical calculations to provide detailed molecular information that offers insight into the substrate specificity of the AlkB family enzymes, thereby uncovering the structural requirements of the ICL formation. In Aim 3 (Lu lab), we will use highly sensitive LC-MS to identify various types of CL in cells under conditions that induce the formation of etheno adducts. In summary, a novel and potentially toxic type of crosslink adduct in DNA will be investigated in test tube, in silico, and in mammalian cells. Investigations of this new type of ICLs should help researchers understand how DNA is damaged during a critical repair process.
