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Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: (a) ClinVar classifications of RAD51D missense variants each year. P/LP = Pathogenic/Likely Pathogenic, B/LB = Benign/Likely Benign, VUS/CON = unknown significance/conflicting reports. ( b-c ) RAD51D forms an obligate heterodimer with XRCC2 to form the ( b ) BCDX2 (PDB: 8GBJ) or ( c ) XRCC3 (X3CDX2) complexes (PDB: 9SVX) through its interaction with RAD51C. ( d ) Schematic of pooled RAD51D variant function assay. RAD51D mutant library cell population is treated with 250 nM olaparib over several passages, selecting for cells with normal RAD51D function. RAD51D variants are quantified within the starting (P0) and final (P2) cell populations by sequencing and scored by their relative enrichment in P2 versus P0.

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Variant Assay, Functional Assay, Mutagenesis, Sequencing

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: Histograms of RAD51D variant abundance, log10(counts/million counts), within pre-selection libraries for each mutagenesis tile. Cutoff line is at 100 cpm (1/10,000).

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Variant Assay, Selection, Mutagenesis

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: ( a ) Distributions of function scores by variant class; nonsense variants in codons 1–304 are perfectly separated from synonymous variants. Vertical lines denote cutoffs for function classifications (LOF, loss-of-function; INT, intermediate; NEU, neutral). ( b ) Per-residue mean missense function scores, grouped by residue surface area exposure status and secondary structure. Unique LOF exposed residues are labeled. ( c ) Variant-to-function heatmap across RAD51D, shaded by function score (white, WT-like; red, null-like) for each mutant amino acid (rows) at each codon position (columns). Variants that do not pass multiple testing correction (lfsr>0.01) are shaded from white to gray, WT residues are boxed, and dark gray denotes missing data. Tracks above heatmaps, from top to bottom: conservation score, protein secondary structure, key domains. Asp (D) or Glu (E) are shown in boldface to highlight stronger effects of these substitutions at some sites. ( d ) Function scores separate pathogenic from benign variants among single-residue variants reported in ClinVar, and provide evidence for missense VUS and those with conflicting reports (all variants plotted SpliceAI score <0.2). Point color denotes variant type, and filled/open points denote statistical significance (i.e. filled: lfsr ≤0.01). ( e ) Precision-recall curve showing classification performance between a set of 211 SNVs with confident P/LP and B/LB variant classification, included in ( a ).

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Variant Assay, Residue, Labeling, Mutagenesis

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: ( a ) Surface representation of RAD51D within the BCDX2 complex (PDB: 8GBJ). Surface residues are colored according to either red (50% pathogenicity) or yellow (20% pathogenicity). Cartoon representations of XRCC2 (purple), RAD51D (light blue), RAD51C (green), and RAD51B (red) are shown consistently throughout. Bound ATP molecules are represented as yellow-orange sticks. ( b ) Specific loss-of-function variants (red, 50% pathogenicity) listed above or (yellow, 20% pathogenicity) listed below the RAD51D schematic are shown with the indicated interaction interfaces indicated. ( c-g ) Binding sites for RAD51C ( c ), ATP site 1 ( d ), XRCC2 ( e ), ssDNA ( f ), and ATP site 2 ( g ).

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: ( a ) Intolerance of negatively charged (i.e. Asp (D) and Glu (E)) residues throughout RAD51D, as measured by the difference between the mean function scores of D/E and non-D/E mutations. Residues with a difference ±0.5 are labeled. ( b ) Mean function scores of residues making contact with other proteins, ATP, or DNA via H-bonds or salt bridges (noted with circles or starbursts, respectively) in the BCDX2 complex (as predicted in PDB: 8GBJ). ( c ) AlphaMissense pathogenicity scores compared to MAVE function scores. AlphaMissense scores were binned as benign, pathogenic, and ambiguous using the published cutoff values. Counts of each plotted category are reported in the table.

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Labeling

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet:

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques:

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: ( a ) Schematic of sister chromatid exchange assay to measure homologous recombination. In this assay, a cassette with a non-functional copy of GFP is integrated into RAD51D CRISPR/Cas9 U2OS cells. This GFP has a unique I-SceI restriction cut site. A DSB can be induced by expression of a plasmid expressing the I-SceI restriction enzyme. GFP expression is restored by use of a homologous template provided on the cassette following homologous recombination. ( b-c ) A plasmid with indicated synonymous or truncation variant was transiently transfected RAD51D CRISPR/Cas9 U2OS cells with a plasmid coding for the I-SceI restriction enzyme. The percentage of GFP+ cells was measured after three days, indicating a recombination event using a GFP fragment on the cassette. The HR proficiency threshold was determined based on comparison with the range of synonymous variants (green bars) to a wild-type RAD51D expressing plasmid (HR >0.75). The threshold for loss of HR function was calculated using the range of truncation variants compared to a wild-type RAD51D expressing plasmid, as <0.6 (indicated in red for the variants). Note that a subset of those variants analyzed here are replotted in as representative variants. The experiment was performed three to seven times with standard deviations plotted. An empty vector was used as a negative control.

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Homologous Recombination, Functional Assay, CRISPR, Expressing, Plasmid Preparation, Variant Assay, Transfection, Comparison, Negative Control

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: (a) The HR proficiency of 70 RAD51D variants was tested using the sister chromatid recombination assay calibrated against synonymous and truncation variants. Loss of HR function was calculated based on the range of truncation variants (indicated in red) as <0.6 (missense LOF in light). HR proficient variants (gray), were determined based on comparison with the range of synonymous variants (green) if they exhibited HR >0.75. Variants with intermediate HR proficiency, in the range of 0.6–0.75, were color-coded in yellow. See for all synonymous and truncation variants tested, and note that a subset of those variants analyzed are replotted here as representative variants. The experiment was performed 4–9 times and plotted as mean values ± s.d. ( b-e ) Olaparib and cisplatin sensitivity of breast/ovarian cancer identified RAD51D variants with reduced HR. Representative images of U2OS cell lines stably expressing WT or the indicated RAD51D variant that were treated with increasing concentrations of Olaparib ( b ) or cisplatin ( d ). ( c & e ) Clonogenic survival assays were quantified by percent colony area and normalized to the area of untreated or vehicle control. Means of 4–12 trials are plotted ± s.d., and drug concentrations with colony area <0.001 are omitted. ( f ) RAD51D variants with reduced HR are expressed. Western blot analysis of U2OS cell lines stably expressing WT or the indicated RAD51D variants. RAD51D protein expression was assessed using an anti-RAD51D antibody, and equal protein loading was assessed using an anti-Tubulin antibody. Note that L4H exhibits reduced protein expression. Experiment performed in triplicate. ( g ) Structures of the RAD51 paralog pentamer, XRCC3 complex, (PDB: 9SVX) and the BCDX2 complex (PDB: 8GBJ). Cartoon representations of XRCC2 (purple), RAD51D (light blue), RAD51C (green), XRCC3 (yellow), RAD51 (orange), and RAD51B (red) are shown consistently throughout. Bound ATP molecules are represented as yellow-orange sticks. ssDNA is shown in orange. ( h-i ) Variants causing complete (red, h ) or intermediate (yellow, i ) deficiency in homologous recombination are highlighted on RAD51D. The interacting surfaces of RAD51C and XRCC2 are outlined with black dashed lines. The contact interfaces are identical between the XRCC3 and BCDX2 complex. ( j-k ) Variants that disrupt RAD51C-RAD51D interactions are shown as red sticks, mapped onto the BCDX2 complex structure.

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Recombination Assay, Comparison, Stable Transfection, Expressing, Variant Assay, Control, Western Blot, Homologous Recombination

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: ( a-b ) Interaction proficiency of RAD51D VUS was measured via ( a ) yeast 2-hybrid (Y2H) of pGAD-RAD51D or its variant, expressed in a GAL4 DNA activating domain expressing plasmid with pGBD-XRCC2 expressed in the GAL4 DNA binding domain expressing plasmid and ( b ) pGAD-RAD51D or its variant, expressed in the pGAD (GAL4 DNA activating domain) plasmid with pGBD-RAD51C (GAL4 DNA binding domain) plasmid with pADH1-RAD51B via yeast 3-hybrid (Y3H). RAD51B serves to stabilize RAD51C protein levels. Empty vectors are used as a negative control. Quantification of yeast growth from three experiments is plotted as mean ± s.d relative to the WT control. Representative images for variants and controls are shown. Variants with <50% interaction are classified as deficient and plotted in red. ( c ) RAD51D variants with reduced interaction with XRCC2 or RAD51C are expressed. Western blot analysis of protein extract from yeast cells in (a) expressing WT RAD51D or the indicated RAD51D variants was assessed using an anti-RAD51D antibody, and equal protein loading was assessed by measuring the amount of Kar2 using an anti-Kar2 antibody. Note that a subset of variants has reduced protein expression relative to WT RAD51D. Experiment performed in triplicate. See for Western blots of all variants. ( d-f ) MAVE function scores vs individual variant assay results of (d) HR activity, (e) XRCC2 binding, and (f) RAD51C binding. Missense variant point shape denotes functional status (LOF/INT/NEU) based upon MAVE result.

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Variant Assay, Expressing, Plasmid Preparation, Binding Assay, Negative Control, Control, Western Blot, Activity Assay, Functional Assay

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: ( a ) Western blot analysis of protein extract from U2OS cells expressing wild-type RAD51D or the indicated RAD51D variants was assessed using an anti-RAD51D antibody, and equal protein loading was assessed using an anti-tubilin antibody. Note that a subset of variants has reduced protein expression relative to WT RAD51D. Experiment performed in triplicate. ( b ) HR analysis of RAD51D variants that are stably expressed in the RAD51D KO cell line used for the clonogenic survival assays shown in .

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Western Blot, Expressing, Stable Transfection

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: Western blot analysis of protein extract from yeast cells expressing wild-type RAD51D or the indicated RAD51D variants was assessed using an anti-RAD51D antibody, and equal protein loading was assessed using an anti-KAR2 antibody. Note that a subset of variants has reduced protein expression relative to WT RAD51D. Experiment performed in triplicate.

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Western Blot, Expressing

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: ( a ) Pull-down analysis of WT BC (RAD51B-His/RAD51C) and DX2 (RAD51D/XRCC2-FLAG) sub-complexes with indicated RAD51D variants using anti-FLAG resin. ( b ) ssDNA binding of BCDX2 paralog complexes reconstituted by mixing WT BC and DX2 sub-complexes with indicated RAD51D variants. ( c ) Pull-down analysis of WT CX3 (RAD51C-His/XRCC3-STREP) and DX2 (RAD51D/XRCC2-FLAG) sub-complexes with indicated RAD51D variants using anti-FLAG resin. ( d ) ATPase analysis of 0.5 μM WT BC and DX2 sub-complexes with indicated RAD51D variants in the presence of ssDNA after 60 min incubation. Pi indicates released inorganic phosphate after hydrolysis. ( e ) ssDNA binding of X3CDX2 paralog complexes reconstituted by mixing WT CX3 and DX2 sub-complexes with indicated RAD51D variants. For ( b, d-e ), results from three independent experiments were plotted as mean values ± s.d.

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Binding Assay, Incubation

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: ( a ) Summary of cellular findings. Schematic of RAD51D (1-328 aa) is shown with the Walker A and B motifs (green), and DNA binding loops (blue) indicated. The variants analyzed are shown above with the results from the cellular studies (MAVE variant-function map; ), RAD51D Y2H interaction with XRCC2 and its Y3H interaction with RAD51C , SCR recombination (HR), as well as olaparib and cisplatin clonogenic survival assays are summarized based on functional score. Loss-of-function is shown in red, an intermediate function is shown in yellow, and wild-type function is indicated in green. ( b ) Summary of biochemical findings. Variants included in biochemical analysis for BCDX2 or XRCC3 (X3CDX2) complex formation, DNA binding, and ATPase restrain from are shown. ( c ) Model for BCDX2 and X3CDX2 function in DSB repair. Upon DSB formation, the 3’ ssDNA end is resected and coated by RPA. The BCDX2 and X3CDX2 complexes facilitate the displacement of RPA and the loading of RAD51 onto ssDNA. This is achieved by the regulation of BC ATPase activity by DX2, which facilitates its binding to ssDNA. CX3 in complex with DX2 then targets RAD51 to ssDNA. The combined functions of the BCDX2 and X3CDX2 complexes are necessary to facilitate RAD51 filament assembly and subsequent RAD51-mediated homology search and strand exchange activities.

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Binding Assay, Variant Assay, Functional Assay, Activity Assay