D. mulleri. D. hexastigma. D. huichole. D. hydei. D. novemaristata. D. ellisoni. D. buzzati. D. nigrospiracula. D. neorepleta. D. stalkeri. D. pachuca. D. longicornis.
Page 1. Dele on at nucleo de posi on G2477, A2478 and C2488 reference DNA sequences. Figure S5.
Page 1. -0.25. 0.00. 0.25. 0.50. 0.75. Log. 10. S e. L a c ta te p = 0.006 p = 0.034. 0. 5. 10. 15. 20. Log. 2. P a ra s ite s. /u. L. 4.00. 4.25. 4.50. 4.75. 5.00. 5.25. Log.
mitochondrial ribosomal protein L51. 13132. -0.837. -0.2181. Yes. 26 DNAJA3. DNAJA3. Entrez,. Source. DnaJ (Hsp40) homolog, subfamily A, member 3.
shp1 shp2 point at individual protoderm cells which are swollen and cause a slightly irregular shape of the embryo outline. (D) to (F), mutant embryos; (G) to (I),.
Frequency distribution (histogram) of cultivars for years of introduction as used in the current study. Year of introduction. F re q u e n c y. 1950. 1960. 1970. 1980.
Page 1. F. M. 7.5. 10.0. 12.5. 15.0. 17.5. F. M. 20. 30. 40. 50 p = 0.005 p = 0.004 p = 0.024. F. M. 50. 75. 100. 125. 150. Figure S2.
ENST00000540450 Synonymous. Het. 42. 11 BATF2. ENST00000534177 Nonsynonymous. Het. 43. 11 SART1. ENST00000542816 Nonsynonymous. Het. 44.
Not Obs Not Obs. Not. Observed. Suppl Fig1C. Page 2. 0. 10. 20. 30. 40. 50. 60. 70. 80. P47 Phox KO. DIO. NAFLD. N o. of C olocaliz ation events / 100 cells.
used to generate standard curves to determine the binding affinity of free RAD51 to each BRCn-DBD protein. B. Quantification of saturation binding analysis of.
A
RAD51 Standards
Amylose Pull-downs
RAD51
RAD51
0.1 0.2 0.4 0.8 0 0.25 0.5 1 3 6 6
BRCn-DBD Standards
BRC4-DBD 1 2
(µg)
4
BRC4-DBD RAD51 RAD51
RAD51
0.1 0.2 0.4 0.8 0 0.25 0.5 1 3 6 6
BRC1-4-DBD 1 2
(µg)
4
BRC1-4-DBD RAD51 RAD51
RAD51
0.1 0.2 0.4 0.8 0 0.25 0.5 1 3 6 6
BRC1-8-DBD 1 2
(µg)
4
BRC1-8-DBD RAD51
1 2 3 4 5 6 7 8 9 101112 13 14 SYPRO Orange
RAD51:BRCn-DBD ratio
B
8 Full-length BRCA2
6
BRC1-8-DBD BRC1-4-DBD
4
BRC4-DBD
2 0 0.0
0.5 1.0 1.5 Total RAD51 (µM)
2.0
Supplementary Figure 1
Supplementary Figure S1. Saturation binding analysis of BRCn-DBD proteins with RAD51. A. SYPRO Orange stained SDS-PAGE gels depicting RAD51 standards (lanes 1-4), amylose pull-downs (lanes 5-10), and BRCn-DBD standards (12-14). Lane 5 is BRCn-DBD alone. Lane 11 is RAD51 alone. Increasing amounts of purified RAD51 (lanes 5-10) were incubated with a fixed concentration of each BRCn-DBD protein for 30 minutes at 37 °C. The amylose beads were then washed and the protein complexes eluted with maltose. The RAD51 and BRCn-DBD standards contained known amounts of protein and were used to generate standard curves to determine the binding affinity of free RAD51 to each BRCn-DBD protein. B. Quantification of saturation binding analysis of BRCn-DBD fusions with RAD51. Increasing amounts of purified RAD51 was incubated with a fixed concentration of the indicated BRCn-DBD proteins. The data were fit to a segmental linear regression (full-length, BRC1-8-DBD, BRC1-4DBD) or a hyperbola (BRC4-DBD) using Prism 6.0. Error bars are S.D. (n=3).
A
EMSA 2XMBP - 5 10 20 40 (nM)
Protein-DNA complex Free DNA
1 2
DNA Strand Exchange
-
RAD51
B
3 4 5
2XMBP 0 2 5 10 20 40 (nM)
Product
*dsDNA 1 2
3 4 5
6 7 8
Supplementary Figure 2
Supplementary Figure S2. DNA binding and DNA strand exchange properties of purified 2XMBP tag protein alone. A. Autoradiogram of EMSA analysis depicting increasing concentrations of the purified 2XMBP tag incubated with radiolabeled 3’ tail DNA. Lane 1 is a no protein control. B. Autoradiogram of DNA strand exchange analysis using the purified 2XMBP tag protein performed as described in Fig. 3. Lane 1 is a no protein control. Lane 2 is RAD51 alone. Lanes 3-8, the reactions were pre-incubated with RPA as described in Fig. 3.
Anti-MBP Wash & Elute Biotinylated 167-mer BRCn-DBD Streptavidin Magnetic Bead
Supplementary Figure 3
Supplementary Figure S3. Binding analysis of BRCn-DBD proteins to biotinylated ssDNA. A. Schematic of biotinylated DNA pull-down assay. Biotinylated ssDNA was incubated with each purified BRCn-DBD or DBD protein for the indicated times, captured on magnetic streptavidin beads, washed, eluted in sample buffer, and analyzed by SDS-PAGE western blotting. B. Representative western blot to detect BRCn-DBD and DBD proteins bound and eluted from biotinylated ssDNA using an antibody directed against the MBP tag.
A
B
1 2 3 4 5 6 7
8
2XMBP-BRC1-8-DBD
Empty Vector
Empty Vector
2XMBP-Full-length BRCA2
9 10
7 21 24 25
Full-length BRCA2
BRC1-8-DBD
Anti-MBP
Anti-MBP
Surviving Fraction
1
Human BRCA2-/-
D Clone 10 Clone 9 Clone 6 Empty Vector
0.1
0.01
0.001 0.00
0.25
0.50
0.75
1.00
Surviving Fraction
C
VC8
1
0.1
Clone 7 Clone 21 Clone 24
0.01
0.001 0.00
Empty Vector
0.25
Mitomycin C (µM)
0.50
0.75
Mitomycin C (µM)
Supplementary Figure 4
1.00
Supplementary Figure S4. Analysis of single cell derived stable clones generated in human BRCA2-/- and hamster VC8 cells. A. Western blot depicting expression levels of 2XMBP tagged full-length BRCA2 in human BRCA2-/- total cell lysates from ten independent stable clones. B. Western blot depicting expression levels of 2XMBP-BRC1-8-DBD in total cell lysates from four independent VC8 stable clones. C. MMC clonogenic survival analysis of three independent human BRCA2-/- stable clones (6, 9, and 10) expressing 2XMBPfull-length BRCA2. D. MMC clonogenic survival analysis of three independent VC8 stable clones (7, 21, and 24) expressing 2XMBP-BRC1-8-DBD.
Full-length
BRC1-8-DBD
BRC5-8-DBD
BRC1-4-DBD
BRC4-DBD
DBD
N-terminus
A
C N C N C N C N C N C N C N Full-length BRCA2 BRCn-DBD proteins MBP PCNA GAPDH
B
Parental BRCA2 -/-
N-terminus
DBD
BRC1-4-DBD BRC5-8-DBD BRC1-8-DBD
Supplementary Figure 5
BRC4-DBD
Full-length BRCA2
Supplementary Figure S5. Intracellular localization of 2XMBP tagged BRCnDBD proteins in human BRCA2-/- cells. A. Biochemical fractionation of cytoplasmic (C) and nuclear (N) extracts from human BRCA2-/- cells expressing the indicated constructs. BRCn-DBD proteins were detected using an MBP antibody. PCNA and GAPDH were used to confirm biochemical partitioning of nuclear and cytoplasmic extracts respectively. B. Representative confocal images using an antibody against MBP (red) to detect intracellular localization of each stably expressed 2XMBP tagged protein in human BRCA2-/- cells. DAPI (blue) was used as a counterstain to identify the nuclei. Magnification (800X).
Human BRCA2-/-
A
Full-length
BRC5-8-DBD BRC1-8-DBD
BRC1-4-DBD
-
DBD BRC4-DBD
kDa
N-terminus
Amylose Pull-downs
BRCA2 BRCn-DBD proteins
250
MBP
RAD51 2
37
4
5
6
7
8
BRC5-8-DBD
BRC1-4-DBD
BRC4-DBD
N-terminus
-
DBD
Total Cell Lysates
B
kDa
3
Full-length
1
BRC1-8-DBD
37
RAD51
Supplementary Figure 6
Supplementary Figure S6. Endogenous RAD51 binding to BRCn-DBD proteins stably expressed in human BRCA2-/- cells. A. Cell extracts were harvested from human BRCA2-/- stable clones expressing the indicated constructs and batch bound to amylose beads in the presence of Benzonase, washed extensively, and eluted with maltose. The BRCn-DBD proteins were detected using an antibody against MBP and the endogenous RAD51 protein was detected by western blotting with an antibody against RAD51. B. Total cell lysates from the cells described in (A) were analyzed by western blotting for RAD51 expression levels.
DAPI
γ-H2AX
RAD51
Empty Vector
N-terminus
DBD
BRC4-DBD
BRC1-4-DBD
BRC5-8-DBD
BRC1-8-DBD
Full-length
Supplementary Figure 7
RAD51 γ-H2AX
Supplementary Figure S7. Ionizing radiation induced RAD51 and γ-H2AX foci in human BRCA2-/- cells expressing BRCn-DBD proteins. Representative confocal images of human BRCA2-/- cells stably expressing the indicated BRCnDBD constructs fixed and stained for RAD51 foci (green) and γ-H2AX foci (red) 8 hours following irradiation (4 Gy). Panels in far left column are DAPI (blue) stained nuclei. Boxed insets depict representative γ-H2AX and RAD51 foci used for co-localization quantification in Figure 6C. Panels in far right column are merged γ-H2AX and RAD51 foci. The images shown in each row contain the same cells.
4 Gy 8 hrs
4 Gy 48 hrs
Empty Vector RAD51
γ-H2AX
N-terminus
DBD
BRC4-DBD
BRC1-4-DBD
BRC5-8-DBD
BRC1-8-DBD
Full-length
Supplementary Figure 8
RAD51
γ-H2AX
Supplementary Figure S8. RAD51 and γ-H2AX foci in human BRCA2-/- cells expressing BRCn-DBD proteins. Representative images of RAD51 foci (green) and γ-H2AX foci (red) in human BRCA2-/- cells stably expressing the indicated BRCn-DBD constructs. Left panels are images taken 8 hours after exposure to 4 Gy of ionizing radiation. Right panels are images taken 48 hours post exposure to 4 Gy of ionizing radiation.
