A Structural Hinge in Eukaryotic MutY Homologues Mediates Catalytic Activity and Rad9–Rad1–Hus1 Checkpoint Complex Interactions

Abstract

The DNA glycosylase MutY homologue (MYH or MUTYH) removes adenines misincorporated opposite 8-oxoguanine as part of the base excision repair pathway. Importantly, defects in human MYH (hMYH) activity cause the inherited colorectal cancer syndrome, MYH-associated polyposis (MAP). A key feature of MYH activity is its coordination with the cell cycle checkpoint via interaction with the Rad9-Rad1-Hus1 (9-1-1) complex. The 9-1-1 complex facilitates cell cycle checkpoint activity and coordinates this activity with ongoing DNA repair. The interdomain connector between the catalytic and 8-oxoG recognition-domains of hMYH (IDC, residues 295-350) is a critical element that maintains interactions with the 9-1-1 complex. We report the first crystal structure of a eukaryotic MutY protein, a fragment of hMYH (residues 65-350) that consists of the catalytic domain and the IDC. Our structure reveals that the IDC adopts a stabilized conformation projecting away from the catalytic domain to form a docking scaffold for 9-1-1. We further examined the role of the IDC using Schizosaccharomyces pombe MYH (SpMyh1) as a model system. In vitro studies of SpMyh1 identified residues I 261 and E 262 of the IDC (equivalent to V 315 and E 316 of the hMYH IDC) as critical for maintaining the MYH/9-1-1 interaction. We determined that the eukaryotic IDC is also required for DNA damage selection and robust enzymatic activity. Our studies also provide the first evidence that disruption of the MYH/9-1-1 interaction diminishes repair of oxidative DNA damage in vivo. Thus, preserving the MYH/9-1-1 interaction contributes significantly to minimizing the mutagenic potential of oxidative DNA damage.

Human MutY homolog induces apoptosis in etoposide-treated HEK293 cells

“…Recently, it has been reported that ATR phosphorylation upon HU or UV treatment was decreased in hMYH-disrupted HEK293 and HaCaT cells (14). ATR is known to phosphorylate Chk1 and Chk2 (14,15), and here, we observed a phosphorylation of Chk2 only in control-transfected cells, but not in siMYH cells following ETP treatment (Fig. 2A).…”

“…Furthermore, in hMYH-disrupted cells, the phosphorylation of ATR and Chk1 is decreased by hydroxyurea (HU) or ultraviolet (UV) treatment (14). The hMYH is known to interact with 9-1-1 (15), and a recent study revealed that it also interacts with ATR and MutS α via the human MutS homolog (hMSH) 6 subunit (14,16).…”

A human MUTYH variant linking colonic polyposis to redox degradation of the [4Fe4S]2+ cluster

“…Indeed, both germline and somatic alterations in other cysteines comprising the [4Fe4S] cluster and the four arginines that participate in hydrogen bonding to the cysteines coordinating the cluster[44] (Supplementary Tables 1 and 2) have been identified and are associated with colorectal as well as other cancers[7]. It is probable that these mutations also result in instability, degradation and dysfunction of the [4Fe4S] cluster secondary to the same mechanisms detailed above.…”

SIRT6 protein deacetylase interacts with MYH DNA glycosylase, APE1 endonuclease, and Rad9–Rad1–Hus1 checkpoint clamp

“…The plasmid containing GST-hMYH(65–350) was derived from pET19b-hMYH(65–350) [11] by PCR amplification using primers listed in Additional file 1: Table S1. The PCR product was digested with BamHI and XhoI and ligated into the digested pGEX-4T-2 (GE Healthcare).…”