The structure and polymerase-recognition mechanism of the crucial adaptor protein AND-1 in the human replisome

Abstract

DNA replication in eukaryotic cells is performed by a multiprotein complex called the replisome, which consists of helicases, polymerases, and adaptor molecules. Human <u>a</u>cidic <u>n</u>ucleoplasmic <u>D</u>NA-binding protein 1 (AND-1), also known as WD repeat and high mobility group (HMG)-box DNA-binding protein 1 (WDHD1), is an adaptor molecule crucial for DNA replication. Although structural information for the AND-1 yeast ortholog is available, the mechanistic details for how human AND-1 protein anchors the lagging-strand DNA polymerase α (pol α) to the DNA helicase complex (<u>C</u>dc45-<u>M</u>CM2-7-<u>G</u>INS, CMG) await elucidation. Here, we report the structures of the N-terminal WD40 and SepB domains of human AND-1, as well as a biochemical analysis of the C-terminal HMG domain. We show that AND-1 exists as a homotrimer mediated by the SepB domain. Mutant study results suggested that a positively charged groove within the SepB domain provides binding sites for pol α. Different from its ortholog protein in budding yeast, human AND-1 is recruited to the CMG complex, mediated by unknown participants other than Go Ichi Ni San. In addition, we show that AND-1 binds to DNA <i>in vitro</i>, using its C-terminal HMG domain. In conclusion, our findings provide important insights into the mechanistic details of human AND-1 function, advancing our understanding of replisome formation during eukaryotic replication.