MUTYH is a clinically important DNA glycosylase that thwarts mutations by initiating base-excision repair at 8-oxoguanine (OG):A lesions. The roles for its [4Fe-4S] cluster and Zn cofactors in DNA repair remain enigmatic. Functional profiling of cancer-associated variants near the [4Fe-4S] cluster revealed that most variations abrogate both retention of the cofactor and enzyme activity. Surprisingly, R241Q and N238S retained the metal cluster and bound substrate DNA tightly but were completely inactive. In contrast, cancer-associated variants near the Zn site had a more modest impact on enzyme activity. We determined several crystal structures of human MUTYH bound to a transition state or product mimic. The structures revealed that Arg241 and Asn238 build an H-bond network connecting the [4Fe-4S] cluster to the catalytic Asp236 that mediates base excision. The structure of the bacterial MutY variant R149Q, along with molecular dynamics simulations of the human enzyme, support a model in which the cofactor functions to position and activate the catalytic Asp. A similar H-bond network mediating interactions between the Zn cofactor site and the [4Fe-4S] cluster were also observed, suggesting that the Zn and [4Fe-4S] site are highly coordinated providing another layer of allosteric control. These results suggest that allosteric cross-talk between the DNA binding [4Fe-4S] cluster and Zn cofactors and the base excision site of MUTYH regulate its DNA repair function.