Figure 2.
Experimental design for NBN variant functional characterization. Parallel NBN variant characterization was done using the engineered NBN−/− HEK293T LP cell line model. First, the 25 variants of interest were fused to EGPF, tagged with a unique barcode sequence, and cloned in the attB-mCherry recombination plasmid. Next, NBN variants were introduced into the attP/attB recombination site in NBN−/− HEK293T LP cells. Cells with successful recombination were identified as mCherry+/BFP− population in flow cytometry. Finally, NBN variant expressing cells were subjected to 2 different types of phenotyping to determine their effect on (1) NBN variant protein stability or (2) NBN variant MMC sensitivity in vitro. NBN protein stability was quantified by the fluorescence intensity of the EGFP-fusion protein and normalized to the co-translationally expressed mCherry fluorescence signal (EGFP:mCherry ratio). Unstable variants resulted in decreased EGFP expression and thus a low EGFP:mCherry ratio, as illustrated by the red-colored histogram. Drug sensitivity was determined by the change in NBN variant frequency after MMC exposure. Damaging variants resulted in the loss of NBN signaling during MMC-induced DNA-damage repair. This led to reduced cell survival and thus under-representation of the respective variants after MMC treatment, which was quantified by targeted sequencing of the barcode region. Finally, protein stability and MMC drug sensitivity were both considered for NBN variant classification. BFP, blue fluorescent protein; EGPF, enhanced green fluorescent protein; MMC, mitomycin C.

Experimental design for NBN variant functional characterization. Parallel NBN variant characterization was done using the engineered NBN−/− HEK293T LP cell line model. First, the 25 variants of interest were fused to EGPF, tagged with a unique barcode sequence, and cloned in the attB-mCherry recombination plasmid. Next, NBN variants were introduced into the attP/attB recombination site in NBN−/− HEK293T LP cells. Cells with successful recombination were identified as mCherry+/BFP− population in flow cytometry. Finally, NBN variant expressing cells were subjected to 2 different types of phenotyping to determine their effect on (1) NBN variant protein stability or (2) NBN variant MMC sensitivity in vitro. NBN protein stability was quantified by the fluorescence intensity of the EGFP-fusion protein and normalized to the co-translationally expressed mCherry fluorescence signal (EGFP:mCherry ratio). Unstable variants resulted in decreased EGFP expression and thus a low EGFP:mCherry ratio, as illustrated by the red-colored histogram. Drug sensitivity was determined by the change in NBN variant frequency after MMC exposure. Damaging variants resulted in the loss of NBN signaling during MMC-induced DNA-damage repair. This led to reduced cell survival and thus under-representation of the respective variants after MMC treatment, which was quantified by targeted sequencing of the barcode region. Finally, protein stability and MMC drug sensitivity were both considered for NBN variant classification. BFP, blue fluorescent protein; EGPF, enhanced green fluorescent protein; MMC, mitomycin C.

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