Name:

E3 ligase activity

Category:

Activity

Property:

Activity

Units:

Unitless

Measurement Technique:

Deep sequencing , Single End Illumina Sequencing , Autoubiquitination

Buffers:

None

Temperature:

Reaction products were treated with exonuclease I (Affymetrix) for 15 min at 37

pH:

None

Protein Concentration:

None

Wavelength:

None

Ionic Strength:

None

Details:

To assay E3 ligase activity, we subjected an allelic series of the BRCA1 N terminus amino acids (2–304) to a phage display assay that selects for protein variants capable of autoubiquitination. We expressed BRCA1 (2–304) variants on the surface of phage and selected for BRCA1 ubiquitination activity over five sequential rounds of selection in the presence of an E1, an E2, and Flag–ubiquitin by capturing phage with anti-Flag beads. Phages that encode active BRCA1 RING variants increase in abundance and those that encode inactive variants decrease in abundance over the multiple rounds of selection.

We used deep sequencing to count each allele in the input phage population after each round. We calculated E3 ligase scores by tracking the changes in the relative abundance of each allele during the selection. For each variant, a linear model was fit by least squares to the log ratios over time. The scores were normalized such that the wild type had a score of one and the mean score for variants with premature termination codons had a score of zero. E3 ligase activity for variants with missense substitutions ranged from completely nonfunctional (scores of zero) to nearly three times higher than wild type.

Additionally, we used cutoff based on the number of input reads to determine the high-confidence data set that would be used for the final HDR predictions. HDR predictions were made only for variants with high-confidence scores in both the E3 ligase and BARD1-binding assays.