Abstract 3779: Mutational analysis of MCPH1 C-terminal tandem BRCT domain reveals residues essential for cell cycle arrest

Abstract

Abstract In this study, the C-terminal tandem BRCT domain of human microcephalin 1 protein (MCPH1) was mutagenized in Saccharomyces cerevisiae yeast to identify residues critical for the cell cycle arrest. Deleterious variants in MCPH1 causing the loss of the protein function are associated with primary microcephaly, characterized by decreased cerebral cortex size and mild mental retardation. The tandem BRCT domain of MCPH1 (amino acids 640-833) recognizes phospho-serine/threonine motifs, including □-H2AX, necessary for DNA damage response and cell cycle regulation. Expression of this region of human MCPH1 tethered to DNA in yeast through the Gal4 DNA binding domain (and not through the interaction with the Gal4 activation domain), results in growth inhibition and a small colony phenotype. To detect the essential residues of the MCPH1 tandem BRCT, mutations were randomly introduced into this region using error-prone polymerase chain reaction. The mutants were then transformed into yeast and transformants showing abrogation of the small colony phenotype were isolated and the specific mutations were assessed by sequencing. Screening revealed 34 missense, 4 nonsense, and 8 frameshift variants, and one large deletion of 72 base pairs. The 34 missense variants were further analyzed in silico by alignment with the BRCA1 tandem BRCT. Out of 34 missense variants, four (T694A, L755P, F756V, and S819P) were identified in two or more colonies. One of the mutated residues (T653) is involved in direct hydrogen bonding with □-H2AX, which is likely disrupted by the amino acid change induced during mutagenesis (T653I). Interestingly, when aligned with BRCA1, 16 (47%) of MCPH1 missense variants matched to a corresponding site in BRCA1 previously reported as a missense variant with a functional impact. MCPH1 residues 653, 756, and 819 all corresponded to amino acids in BRCA1 known for their likely pathogenicity. Thus, this study identifies functional determinants in MCPH1 C-terminal domain potentially involved in cell cycle regulation. Taken together, our results suggest that variants in BRCT domain containing proteins, can be clinically annotated by making comparative alignments with the conserved residues of BRCA1 BRCT. Structural inference using BRCT paralogs might aid in the clinical annotation for variants lacking genetic data. Citation Format: Volha A. Golubeva, Nicholas T. Woods, Alvaro N.A. Monteiro. Mutational analysis of MCPH1 C-terminal tandem BRCT domain reveals residues essential for cell cycle arrest. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3779. doi:10.1158/1538-7445.AM2015-3779