| Antibodies |
|
|
|
| Mouse anti-Beta-actin |
Santa Cruz |
Cat #: sc-47778; RRID: AB_2714189 |
| Mouse anti-Flag M5 |
Sigma Aldrich Inc |
Cat #: F4042; RRID: AB_439686 |
| Rabbit anti-Gamma-H2AX |
Cell Signaling Technology (CST) |
Cat #: 2577; RRID: AB_2118010 |
| Rabbit anti-GAPDH |
CST |
Cat #: 5174; RRID: AB_10622025 |
| Rabbit anti-GFP |
Biomol |
Cat #: 600-401-215S; RRID: AB_2612814 |
| Rabbit anti-Histone 3 |
CST |
Cat #: 4499; RRID: AB_10544537 |
| Mouse anti-KU70 |
Santa Cruz |
Cat #: sc-17789; RRID: AB_628454 |
| Rabbit anti-MRE11 |
CST |
Cat #: 4895; RRID: AB_2145100 |
| Mouse anti-PAR |
Abcam |
Cat #: 14459; RRID: AB_301239 |
| Rabbit anti-PAR |
Millipore Sigma |
Cat #: MAB3192; RRID: AB_94743 |
| Rabbit anti-PARP |
CST |
Cat #: 9532; RRID: AB_659884 |
| Rabbit anti-RAD51 |
Santa Cruz |
Cat #: sc-8349; RRID: AB_2253533 |
| Goat anti-RAD52 |
A gift from Dr. Jiri Lukas’s lab |
(Ochs et al., 2016) |
| Rabbit anti-TurboGFP (tGFP) |
Thermo Fisher |
Cat #: PA5-22688; RRID: AB_2540616 |
| Rabbit anti-XRCC1 |
CST |
Cat #: 2735; RRID: AB_2218471 |
| Mouse anti-XRCC1 |
Abcam |
Cat #: Ab1838; RRID: AB_302636 |
| Mouse anti-LIG3α |
Thermo Fisher Scientific Inc. |
Cat #: PA5-21480; RRID: AB_11153448 |
|
| Chemicals, Peptides, and Recombinant Proteins |
|
|
|
| CPT |
VWR |
101757-582 |
| etoposide |
VWR |
ss419-42-0 |
| RAD52 inhibitor, CD1 |
ChemDiv Inc. |
C267-0065 |
| PARP inhibitor, niraparib |
SELLECK |
S7625 |
| PARP inhibitor, olaparib |
SELLECK |
S1060 |
| PARG inhibitor |
Millipore Sigma |
118415 |
| methyl methanesulfonate (MMS) |
Sigma-Aldrich Inc |
129925-5G |
| H2O2
|
Fisher Scientific |
H325-100 |
| aphidicolin |
Sigma-Aldrich Inc |
A4487 |
| nocodazole |
CST |
2190 |
| DAPI |
Vector Laboratory |
H-1200 |
|
| Critical Commercial Assays |
|
|
|
| CellTiter-Glo Luminescent Cell Viability Assay Kit |
Promega Corp |
G9241 |
| Chromatin Extraction Kit |
Abcam |
ab117152 |
| Single cell electrophoresis (comet assay) Kit |
Trivigen |
4250-050-K |
| Duolink In Situ Orange Starter (Mouse/Rabbit) Kit |
Sigma-Aldrich Inc |
DUO92102-1KT |
| PARP Inhibition Assay Kit |
Trevigen |
4690-096-K |
|
| Experimental Models: Cell Lines |
|
|
|
| DT40 WT, Rad52-deficient |
Dr. Shinichi Takeda’s Lab |
(Yamaguchi-Iwai et al., 1998) |
| DT40 Rad52/Ku70-deficient |
This study |
N/A |
| DT40 Rad54, Ku70, Rad54/Ku70-deficient |
Dr. Shinichi Takeda’s Lab |
(Takata et al., 1998) |
| DT40 Atm, Atm/Ku70-deficient |
Dr. Shinichi Takeda’s Lab |
(Morrison et al., 2000) |
| DT40 Parp1 or Parp11/Ku70 deficient |
Dr. Shinichi Takeda’s Lab |
(Hochegger et al., 2006) |
| DT40 Polq (theta)-deficient |
Dr. Shinichi Takeda’s Lab |
(Kohzaki et al., 2010) |
| MEF WT and Ku70-deficient |
Dr. David Chen’s Lab |
(Li et al., 1998) |
| MEF Parp1-deficient |
Dr. Zhaoqi Wang’s Lab |
(Wang et al., 1995) |
| MEF Rad54-deficient |
Dr. George Iliakis’s Lab with Dr. Kevin Mill’s permission |
(Mills et al., 2004) |
| MEF Xrcc1-deficient |
Dr. Li Lan’s lab |
(Wei et al., 2013) |
MEF Rad52-deficient or Rad52 mutants including Rad52R55A
|
In this study |
N/A |
|
Rad52Y104D and Rad52Y104F
|
| HEK293T |
ATCC |
ACS-4500 |
| U2OS |
ATCC |
HTB96 |
|
| Oligonucleotides |
|
|
|
| KO cXrcc6-Ex3, F: 5′-CACCAAGCGTTGTGTTCTACGGCA-3′ |
IDT |
N/A |
| KO cXrcc6-Ex3, R: 5′-AAACTGCCGTAGAACACAACGCTT-3′ |
IDT |
N/A |
| KO cXrcc6-Ex4, F: 5′-CACCGGCATGAGGGTTATCCTCGT-3′ |
IDT |
N/A |
| KO cXrcc6-Ex4, R: 5′-AAACGACTGATTACCTTGTGACCC-3′ |
IDT |
N/A |
| KO mRad52-Ex3, F: 5′-CACCATTATAGAGCCAGTATACAG-3′ |
IDT |
N/A |
| KO mRad52-Ex3, F: 5′-CACCATTATAGAGCCAGTATACAG-3′ |
IDT |
N/A |
| KO mRad52-Ex3, R: 5′-AAACCTGTATACTGGCTCTATAAT-3′ |
IDT |
N/A |
| KO mRad52-Ex5,F: 5′-CACCAACAATGGCAAGTTCTACGT-3′ |
IDT |
N/A |
| KO mRad52-Ex5, R: 5′-CACCTTAGTTAAATCCACATCAAG-3′ |
IDT |
N/A |
| Primers for mutations of mRad52 R55A, Y104F and Y104D are as described in Table S1
|
IDT |
N/A |
| A biotin-labeled DNA oligo Top: GGCCGCACGCGTCCACCATGGGGTAC AA [BioTEGi] GCACGCGTCCACCATGGGGTACAA; |
Eurofins |
N/A |
| A biotin-labeled DNA oligo Bottom: GTAGTTGTACCCCATGGTGGACGCGTGCTTGTACCCCATGGTGGACGCGTGC |
Eurofins |
N/A |
|
| Recombinant DNA |
|
|
|
| pX330-U6-Chimeric_BB-CBh-hSpCas9 |
Addgene |
(Cong et al., 2013) |
| p3XFLAG-CMV |
Sigma-Aldrich Inc |
E7533 |
| 3xFLAG-Rad52 |
In this study |
N/A |
| 3xFLAG-Rad52 R55A
|
In this study |
N/A |
| 3xFLAG-Rad52Y104F
|
In this study |
N/A |
| 3xFLAG-Rad52Y104D
|
In this study |
N/A |
|
| Software and Algorithms |
|
|
|
| ImageJ |
NIH |
https://imagej.nih.gov/ |
| Comet Score analysis software |
TriTek Corp |
N/A |
| SoftWorx 5.5 |
Applied Precision |
N/A |
|
| Other |
|
|
|
|
mParp2 siRNA 5′-CTACCTTATTCAGCTGTTA-3′ |
Santa Cruz |
N/A |
|
mParp2 siRNA 5′-GTTGAAGCGTGCAATGAAT-3′ |
Santa Cruz |
N/A |
|
mParp2 siRNA 5′-CCATCACAGGTTATATGTT-3′ |
Santa Cruz |
N/A |
|
hRad52 siRNA 5′-GGAGUGACUCAAGAAUUAATT-3′ |
QIAGEN |
SI02629865 |
|
hRad52 siRNA 5′-GGCCCAGAAUACAUAAGUATT-3′ |
QIAGEN |
SI03020794 |
