Fig 2: Comparative Analysis of the Proteomes of XRCC4 Family Proteins isolated from Soluble Chromatin. Interactome analysis using FLAG-tagged PAXX, -XLF, -XRCC4 and -DNA-PKcs as bait from the benzonase-treated soluble chromatin fraction of HEK293F cells was performed using Cytoscape. Proteins highlighted by shaded yellow boxes indicate proteins which interact with the indicated bait protein e.g. PAXX, XLF, XRCC4 or DNA-PKcs. Shaded grey boxes depict proteins identified in combined proteomes of XRCC4 family proteins and DNA-PKcs (PRKDC)

Fig 3: XRCC4 family proteins stimulate gap filling synthesis activity of Pol λ. a Gap filling activity of Pol λ-WT and a catalytically inactive Pol λ-D427A/D429A/D490A mutant. b PAXX, XLF and XRCC4 stimulate gap-filling synthesis activity of Pol λ with an IRDye® 700-labelled 5nt-gapped dsDNA (33-mer) substrate. c As described in Panel B, except that some reactions also contained either FLAG-PAXX or –XLF alone d Gap filling synthesis assays were performed as described in Panel B with Pol λ immunoprecipitated from RPE-1 PAXX+/+ or PAXX KO cells incubated with or without XLF or XRCC4 siRNA

Fig 4: Pol λ, PAXX and XLF function in common and parallel pathways. a Immunoblot analysis of control- or Pol λ siRNA-depleted U2OS PAXX KO, XLF KO and PAXX/XLF DKO cells. WCL were resolved by SDS-PAGE and indicated proteins detected by immunoblotting. b Clonogenic survival assays following IR (0-4 Gy) for U2OS WT, -PAXX KO, -XLF KO and -PAXX/XLF DKO cells with or without depletion of Pol λ Mean and SD from three independent experiments are shown. Statistical analysis was performed using a two-tailed paired t-test to compare cells incubated with Pol λ siRNA with control siRNA: 1 Gy - WT p = 0.91, PAXX KO p = 0.38, XLF KO p = 0.10, PAXX/XLF DKO p = 0.10; 2 Gy - WT p = 0.0003, PAXX KO p = 0.0007, XLF KO p = 0.36, PAXX/XLF DKO p = 0.82; 4 Gy - WT = 0.02, PAXX KO = 0.002, XLF KO p = not determined, PAXX/XLF DKO p = not determined. c Cartoon showing a model for regulation of Pol λ by XRCC4 family proteins. At DSBs that are positioned proximal to a Pol λ substrate gap XRCC4 family proteins strongly interact with Ku heterodimers via their C-terminal regions; their head domains promote gap filling synthesis activity via comparatively weakly binding to the 8 kDa domain of Pol λ, which interacts with the 5′ end of the gap. Pol λ strongly interacts with Ku heterodimers via its N-terminal BRCT domain

Fig 5: Role of XRCC4 family members in the recruitment of Pol λ to laser microirradiation-induced DNA damage sites. a Upper, Schematic figure showing N-terminal EGFP- and mCherry-Pol λ fusion proteins; Lower, Representative immunofluorescence images showing that N-terminal EGFP- and mCherry-Pol λ fusion proteins are localised to nuclei in U2OS cells. b Recruitment of N-terminal EGFP-Pol λ to laser-induced DNA damage sites in U2OS cells. c Time course of N-EGFP-Pol λ recruitment to laser-induced DNA damage sites in U2OS cells. Data shown are the mean and SEM from 16 individual cells. d Immunoblot analysis of N-EGFP-Pol λ expressing U2OS cells deficient in PAXX, XLF or XRCC4 generated by CRISPR-Cas9. WCL were resolved by SDS-PAGE and the indicated proteins detected by immunoblotting. e Localisation of N-mCherry-Pol λ in U2OS WT, PAXX-, XLF- and XRCC4-deficient cells. Representative immunofluorescence images showing that N-terminal mCherry-Pol λ fusion protein localises to nuclei in PAXX-, XLF- or XRCC4-deficient U2OS cells. Cells were co-stained with DAPI or dynamin-2, a perinuclear-enriched protein. f Time course of N-EGFP-Pol λ recruitment to laser-induced DNA damage sites in U2OS-WT cells and cells deficient in PAXX, XLF or XRCC4. Data shown are the mean and SEM from WT, PAXX, XLF and XRCC4 knockout cells. Graphs shown are for the following cell numbers: WT: 8 cells; PAXX KO, 10 cells; XLF KO 17 cells; XRCC4 KO 18 cells. g Time course of N-EGFP-FLAG-Ku70 recruitment to laser-induced DNA damage sites in U2OS-WT cells and PAXX KO cells. Data shown are the mean and SEM from WT and PAXX knockout cells. Graphs shown are for the following cell numbers: WT: 17 cells; PAXX KO, 19 cells