Proline variants in the BRCA1 coiled-coil domain disrupt folding and binding to PALB2.

Abstract

Inherited mutations in the genes coding for the tumor suppressor proteins BRCA1 and PALB2 can lead to increased risk of breast and ovarian cancer. Upon DNA damage, these two proteins form a complex to promote double-stranded break repair via homologous recombination. Missense mutations in either BRCA1 or PALB2 that disrupt this important interaction result in loss of effective DNA damage repair and are associated with breast tumorigenesis. However, the overwhelming majority of missense mutations found in the binding domains of these two genes remain classified as variants of unknown significance. Here we report an in vitro assay for assessing the effect of variants of unknown significance on the heterodimerization of PALB2 and BRCA1 that recapitulates the effect of the known deleterious mutations. We apply the assay to several variants of unknown significance in BRCA1 which reveals other mutations in this region that also disrupt binding, including a mutation of a residue not predicted to directly interact with PALB2. Structural analysis indicates that all BRCA1 mutations to proline tested disrupt α-helix formation and therefore are not well tolerated even when located at positions outside of the PALB2-binding interface. This assay and the structural hypothesis described will be helpful for assessing risk for variants identified in the future in the BRCA1/PALB2 interaction domains.