| dc.description.abstract | Uracil, a common base in RNA, may arise in cellular DNA by cytosine deamination or during replication through the misincorporation of deoxyuridine monophosphate instead of thymidine monophosphate. Repair of uracil is initiated by base excision by an uracil-DNA glycosylase (UDG), where the most important in human cells is hUNG, followed by removal of the abasic site, insertion of the correct base and ligation, all steps are called the base excision repair pathway. In activated B cells, uracil residues introduced by activation-induced cytidine deaminase (AID) are important for immunoglobulin gene diversification, where excision of uracil by hUNG is the key role in introducing the double-strand breaks necessary for class switch recombination (CSR) or point mutations for somatic hypermutation (SHM). We have analyzed the activity of hUNG for two substrates mimicking the intermediates during CSR, bubble and R-loop U-DNA, and tested our hypothesis whether hUNG could incise their DNA strand in addition to excision of uracil. To study hUNG activity, we produced a pure fraction of wild type hUNG, and successfully purified two mutant hUNG proteins, Ser169Ala and Ser270Ala, to investigate the role of these amino acid residues in uracil excision and DNA incision at uracil in DNA. We have determined the kinetic parameters of wild type hUNG for uracil excision in bubble and R-loop DNA, and of hUNG S169A and hUNG S270A for uracil excision in bubble U-DNA. | |