MutY Homologue is a Caretaker Gene and an Adaptor of the DNA-Damage Response Pathway: Transient Multiprotein-DNA Complexes Lead the Way during Base Excision Repair

Abstract

MutY homologue (MYH), a base excision repair (BER) glycosylase, initiates repair of one of the most common oxidative DNA lesions, 7,8-dihydro-8-oxoguanine (8-oxoG). Specifically, MYH excises adenine bases that are frequently misincorporated opposite 8-oxoG during DNA replication, thereby preventing G:C → T:A mutations. Importantly, biallelic mutations in MYH were recently linked to an inherited colorectal cancer syndrome, MYH-associated polyposis, marking the first known causal relationship between a BER defect and cancer. Examination of the genetic pathway to tumorigenesis revealed that defective MYH allows downstream mutations in tumor suppressors and oncogenes to go unrepaired. Research on MYH has largely focused on the catalytic mechanism by which MYH repairs DNA. However, MYH has also been proposed to act as an adaptor protein in the DNA damage response pathway mediating signals of DNA damage to sensor proteins, such as the Rad9-Rad1-Hus1 (9-1-1) complex, which then transduce signals to effector proteins to commence DNA repair, cell cycle arrest, or apoptosis. Our laboratory is interested in the protein-protein interactions that link MYH to the DNA damage checkpoint response pathway. Our x-ray crystallography structure of the catalytic domain and interdomain connector (IDC) of human MYH reveals that the IDC forms a docking scaffold to facilitate transient protein interactions with the essential BER enzyme, AP endonuclease 1 (APE1), and 9-1-1. Although APE1 and 9-1-1 both bind to the IDC of hMYH, their interactions are different; a single point mutation of a conserved residue in the IDC attenuates the MYH/9-1-1 interaction but has no effect on the APE1 interaction. Finally, we demonstrate that disruption of the MYH/9-1-1 interaction diminishes repair of oxidative damage both in vitro and in vivo. Via nuclear magnetic resonance titration experiments, we show that MYH binds to the DNA-binding site of APE1, suggesting a direct handoff of BER DNA intermediates between the two enzymes. Unexpectedly, 9-1-1 stabilizes the MYH/APE1 interaction, and we propose that 9-1-1 assembles MYH and APE1 into a multiprotein complex on DNA lesions to enhance the efficiency of BER. Our research shows that the IDC of hMYH mediates essential interactions with 9-1-1 and APE1 in oxidative DNA damage repair.