Mechanisms of mammalian DNA Repair

DNA repair prevents cancer and early aging and is essential for adaptive immunity. Our objective is to obtain detailed understanding of the mechanism of base excision repair (BER) and direct base damage repair by human AlkB homologs. In addition, we will now expand our research into possible epigenetic gene regulation by some AlkB homologs. BER is a template-dependent multistep process initiated by a DNA glycosylase. The UNG-gene encodes uracil-DNA glycosylases UNG1 located in mitochondria and UNG2 in nuc lei. UNG-mutations are associated with immune deficiency and lymphoma. UNG2 engages in multiprotein complexes that carry out complete BER. We will determine the composition and function of different complexes. They seem to vary depending on type of damage and cell cycle status. We will also continue our studies on the significance of UNG2 for antibody production in the immune system. For this purpose we have now backcrossed our Ung-knockout mice 8 generations to ensure that the genotype is comparable with that of wild type, except for the Ung-gene. Repair of mtDNA has been little studied. We have found that UNG1 is the only uracil-DNA glycosylase in mitochondria. We will continue our comparative work of BER in mitochondria and nuclei to understand mechani sms and mutation frequencies. Direct DNA-base repair by oxidative dealkylases is a template-independent, one-step mechanism carried out by homologs of the E. coli protein AlkB. Eight mammalian AlkB homologs (ABH1-8) have been identified, but only two sign ificantly explored. At least two homologs also repairs RNA. We will examine the function of ABH1,2,3 and 8 in repair or natural modification of DNA, RNA (e.g. tRNA) and possibly methylated histones, and their biomedical significance. We are presently impl ementing mass spectrometry assays for analysis of methylated proteins to study possible epigenetic functions of AlkB homologs. We will use both in vitro systems, human cell models and mouse models for this purpose.