| Escherichia coli MutY is a 39 kDa adenine glycosylase involved in base excision repair (BER) of DNA damage. The MutY enzymes from E. coli, mice and humans are comprised of two domains. The N-terminal catalytic domain contains several motifs involved in DNA damage recognition and an [4Fe-4S]+2 cluster ligated by a sequence of cysteine residues not found in other iron-sulfur cluster containing enzyme classes. The unique spacing of cysteine residues allows for the formation of a loop between the first two ligating cysteine residues. This loop has been found to be involved in substrate recognition and catalysis. Work in this paper reveals a role in substrate recognition for the C-terminal domain.; Studies using structural biology and biochemical techniques on the [4Fe-4S] +2 cluster region elucidate both the size and ligand dependence of this loop and cluster region. The data reveal a faster kinetic rate for a five-membered FCL loop as opposed to the natural six-residue loop found in E. coli. However histidine coordination at position 199 shows a partial loss of iron coordination, yet, the kinetic behavior is unchanged from wild type MutY. X-ray crystallography reveals no deviations from wild type MutY.; Familial adenomatous polyposis (FAP) is a form of colon cancer linked to an inherited defect in the APC tumor suppressor gene. Studies on Family N, has revealed FAP-like symptoms with no inherited defect in APC. Sequencing of the human MutY gene (hMYH) revealed two biallelic mutations in the affected members of this family. These mutations were cloned into MutY enzymes from several species, and their activity was tested both in vivo and in vitro. Complementation assays revealed an inability of these mutants to reduce the cellular mutation rate in an E. coli strain lacking MutY in vivo. In addition, a kinetic analysis of the mutants in E. coli MutY reveal a reduced rate of chemistry for both mutants in vitro.; These studies on the BER adenine glycosylase MutY shed light on the role of the C-terminal domain and [4Fe-4S]+2 regions of the enzyme. In addition, the data presented provide the first link between deficiencies in hMYH and disease. |
