Supplemental Figure Legends
Supplemental Figure 1. c.1432A>G homozygous mutation of mtPAP in affected
members of RS63 family. Displayed is a section of exon 9 of the MTPAP gene where the c.1432A>G mutation is located. The mother, RS63-4, -5, and -6 are carriers (heterozygotes) of the mutation while RS63-3 and -7 are homozygous for the mutation.
Supplemental Figure 2. Representative images of neutral comet assay in
RS63-3 and -7. Representative images from the neutral comet assay post-15 Gy indicate a delay in repair in A-T, RS63-3, and -7 LCLs. All cells display DSBs, indicated by long comet tails, at 30 minutes post-IR. Transfection of WT mtPAP in to
RS63-3 and -7 cells rescues the DSB repair defect and returns tail lengths at 5-hours post-IR to WT-like levels. +CTL indicates transfection of empty vector into RS63-3 or
-7 cells.
Supplemental Figure 3. Increased 53BP1 foci and decreased BRCA1 and
RAD51 foci post-IR in RS63-3 and -7. A. 53BP1 irradiation induced foci (IRIF) kinetics indicating increased foci at 1-hour post-2 Gy and persistent foci at 4-hours in RS63-3 and -7. B. Foci kinetics for BRCA1 displayed reduced BRCA1 foci accumulation in RS63-3 and -7 8-hours post-12 Gy. D. RAD51 foci levels were assess at 8 hrs post-12 Gy, the time when BRCA1 foci levels were also decreased, as a second biomarker of HR repair and were reduced in RS63-3 and -7. For A-C, results are presented as the % cells positive for 4 or more foci/nucleus and an asterisk denotes statistical significance at the p<0.05 level.

Supplemental Figure 4. Cell cycle distribution is normal in RS63-3 and -7
before and after IR. Representative cell cycle histograms are shown for WT, A-T,
RS63-3 and RS63-7 LCLs after FACS analysis of propidium iodide (PI) stained cells and indicate WT-like cell cycle distribution at no-IR, 1-hr and 24-hrs post-10 Gy in
RS63-3 and -7. X-axes for the histograms displayed are FL2-A (PI staining intensity), are given in relative fluorescent units, and represent DNA content levels.
Supplemental Figure 5. S-Phase cell cycle checkpoint is activated in RS63-3
and -7 in a WT-like manner. The integrity of the intra-S phase checkpoint was assessed with BrdU staining post-10 Gy. RS63-3, -7 and WT LCLs displayed a reduction in cells staining positive for BrdU post-IR, indicating DNA synthesis arrest in these cells. A-T LCLs, however, display no appreciable reductions in cells undergoing DNA synthesis. Shown are representative scatter plots. Y-axes are relative fluorescence intensities for BrdU staining and x-axes represent DNA content, indicated by APC staining for DNA content.
Supplemental Figure 6. G2/M checkpoint is activated in RS63-3 and -7 in a
WT-like manner. The G2/M checkpoint was assessed by H3pS10 staining of cells undergoing mitosis. Representative images from FACS analysis of H3pS10 staining are shown. WT-like reduction of H3pS10 in RS63-3 and -7 indicates normal G2/M arrest of cells 1-hour post-10 Gy.
Supplemental Figure 7. Transfection of WT mtPAP rescued increased maximal
mitochondrial respiration phenotype in RS63-3 and -7. Mitochondrial bioenergetics were assessed using the XF24 Extracellular Flux Analyzer and representative data of the oxygen consumption ratio (OCR) after addition of the

respiratory chain decoupler, 0.75  M FCCP, are displayed. Control (-) transfected
RS63-3 and -7 cells displayed increased maximal mitochondrial respiration, while
WT mtPAP cDNA (+) transfection partially rescued maximal respiration levels to near WT-like levels and demonstrated a link between MTPAP mutation and the mitochondrial respiration phenotype observed. Inset in the upper left corner of the figure is a depiction of the curve assessed by the mitochondrial bioenergetics assay.
For clarity of presentation, the timeframe outlined by the grey box, which signifies maximal mitochondrial respiration levels after addition of the respiratory chain decoupler FCCP, is being displayed as a bar graph in this figure. Values presented are the average respiration levels of the three measurements taken at measurement time 7-9. Error bars are +/- 1 standard deviation.
Supplemental Figure 8. Relative increase in reactive oxygen species (ROS)
levels (+IR/-IR) in RS63-3 and -7. A. Marginally increased basal levels of ROS levels were observed in RS63-3 and -7. Displayed is the relative increase (+IR - -IR) in ROS levels and indicates that, regardless of basal levels of ROS, the relative increase in ROS levels post-10 Gy is significantly increased in RS63-3 and -7.
Asterisks denote statistical significance at the p<0.05 level. B. DHE is a cell permeable dye that preferentially detects superoxide levels in living cells. Increased
DHE staining was observed in RS63-3 and -7 without irradiation (-IR) and these cells demonstrated increased superoxide levels post-10 Gy (+IR), while there was not a significant IR-induced response in WT or A-T cells. The ‘WT’ sample represents the averaged value of three different WT-LCLs. DHE fluorescent levels were analyzed by FACS (FL-2) and the results are presented as the percentage of

cells staining positive (% Pos) for larger than WT-levels of superoxide. An asterisk denotes statistical significance at the p<0.05 level comparing –IR to +IR conditions.
Two asterisks denotes statistical significance at the p<0.05 level comparing the –IR
WT condition to the –IR RS63-3 and -7 conditions C. RS63-3 and -7 were transfected with control (CTL) or WT mtPAP cDNA (mtPAP) and superoxide levels were assessed with the DHE dye 2 days later. Transfection of WT mtPAP cDNA reduced superoxide levels to near WT-like levels while the control plasmid gave no improvement for superoxide levels. Further, the IR-induced increase in superoxide levels observed in RS63-3 and -7 were abrogated by WT mtPAP cDNA. DHE fluorescent levels were analyzed by FACS (FL-2) and the results are presented as the percentage of cells staining positive (% Pos) for larger than WT-levels of superoxide.
An asterisk denotes statistical significance at the p<0.05 level.
Supplemental Figure 9. Increased induction of post-IR double strand breaks was abrogated in RS63-3 and 7 with  -LA. The initial induction of DNA breaks was assessed by the NCA after pre-IR treatment with  -LA or DMSO control post-2 and -5 Gy. The initial levels of DNA breaks were reduced to WT levels in RS63-3
(blue arrow) and -7 (orange arrow) by pre-IR treatment with  -LA. Values are presented as the tail moments (TM) post-2 and -5 Gy. Asterisks indicate significance at the p<0.05 level.
Supplemental Figure 10. Similar to alpha-lipoic acid, n-acetylcysteine rescues
defective DDR phenotype in RS63-3 and -7. A. WT, RS63-3 and -7 LCLs were pretreated overnight with 10 and 100  M NAC and initial DNA damage induced by 2 Gy was assessed by measuring  -H2AX foci levels at 1 hour post-IR. NAC pre-treatment

rescued increased  -H2AX foci levels in RS63-3 and -7 while NAC had no impact on foci levels in the WT-LCL. Results are presented as the percentage of cells positive
(% Pos) for 4 or more foci/nucleus B. DNA repair efficacy was assessed 5 hrs post-
15 Gy by the neutral comet assay after pre-treating cells with 10  M NAC. Reduced
DNA DSB repair was rescued in RS63-3 and -7 by pre-treatment with NAC while
NAC had no impact on WT3 repair levels. Results are presented as percent repair
(% Repair), which are the tail moments without irradiation (TM
0hrs
) divided by the residual tail moments (i.e. unrepaired DNA) at 5 hrs post-IR (TM
5hrs
). C. Clonogenic survival was assessed using the CSA post-1 Gy, and pre-treatment with 10  M NAC rescued the reduced levels of clonogenic survival in RS63-3 and -7 to WT-like levels.
Results are presented as the survival fraction percentage (% SF) and the WT and AT values are the ranges established by Sun et al. (4). For A-C, an asterisk represents statistical significance at the p<0.05 level when using the Student’s t-test to compare
NAC treated conditions to the control (H2O).