REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
anti-RPA70	Abcam	Cat# ab79398; RRID: AB_1603759
anti-BRCA2	Merck	Cat# OP95-100UG; RRID: AB_213443
anti-BRCA2 (aa 188-563)	Abcam	Cat# ab97; RRID: AB_296572
anti-BRCA2 (aa 1651-1821)	Oncogene (now Millipore)	Cat# OP95; RRID: AB_2067762
Anti-GFP	Roche	Cat# 11814460001; RRID: AB_390913
Anti-FLAG M2	Sigma-Aldrich	Cat# F1804-50UG; RRID: AB_262044
anti-RAD51	Abcam	Cat# ab213; RRID: AB_302856
Goat anti-Mouse IgG (H+L) Highly Cross-AdsorbedSecondary Antibody, Alexa Fluor Plus 800	Thermo Fisher	Cat# A32730; RRID: AB_2633279
Goat anti-Rabbit IgG (H+L) Highly Cross-AdsorbedSecondary Antibody, Alexa Fluor Plus 680	Thermo Fisher	Cat# A32734; RRID: AB_2633283
Bacterial and virus strains
E. coli BLR(DE3)pLysS	Novagen	Cat# 69956-3
E. coli DH5alpha	NEB	Cat# C2987H
Chemicals, peptides, and recombinant proteins
Ampicillin, Sodium Salt	Merck	Cat# 171254
Chloramphenicol	Merck	Cat# 220551
IPTG, Dioxane-Free, High Purity	Merck	Cat# 420322
Halt™Protease and Phosphatase Inhibitor Cocktail	Thermo Fisher	Cat# 78440
anti-FLAG M2 resin	Merck	Cat# A2220
Econo-Pac gravity flow column	BioRad	Cat# 7321010
SP Sepharose Big Beads, 1 L	Cytiva	Cat# 17065703
Bio-Rad Protein Assay Dye Reagent Concentrate	BioRad	Cat# 5000006
Insect-XPRESS™Protein-free Insect Cell Medium	Lonza	Cat# BELN12-730Q
Sf-900™II SFM	Thermo Fisher	Cat# 10902088
DENARASE, 500 kU	c-LEcta	Cat# 20804-500k
Superdex 200 Increase GL10/300 column	GE Healthcare	Cat# 28-9909-44
Slide-A-Lyzer MINI dialysis units	Thermo Fisher	Cat# 96570
VivaSpin 500 centrifugal concentrator	Sartorius	Cat# VS0121
MaxiGeBaFlex dialysis tube	Generon	Cat# D045
β-mercaptoethanol	Sigma-Aldrich	Cat# 101458612
guanidine hydrochloride	Sigma-Aldrich	Cat# 50940
TCEP (tris(2-carboxyethyl)phosphine)	Sigma-Aldrich	Cat# 646547
StrepTrap HP column (1 ml)	Cytiva	Cat# 28907546
HiTrap Heparin HP column (5 ml)	Cytiva	Cat# 17040703
HisTrap HP column (1 ml)	Cytiva	Cat# 17524701
HiTrap Q HP column (1ml)	Cytiva	Cat# 17115301
HiTrap SP HP column (1ml)	Cytiva	Cat# 29051324
100 kDa MWCO Amicon centrifugal filter	Millipore	Cat# Z648043-24EA
HiTrap Heparin HP column (1 ml)	Cytiva	Cat# 17040601
HiTrap Q FF column (5 ml)	Cytiva	Cat# 17515601
RESOURCE Q anion exchange chromatography column	Cytiva	Cat# 17117701
Mono Q 5/50 GL	Cytiva	Cat# 17516601
Alexa Fluor 647 C2 maleimide	Thermo Fisher	Cat# A20347
Vivaspin centrifugal concentrator (30 kDa MWCO)	Merck	Cat# Z614246-25EA
Zeba Spin Desalting Columns, 7K MWCO, 0.5 mL	Thermo Fisher	Cat# 89882
Streptavidin Mag Sepharose	Cytiva	Cat# 28985799
Sodium hypochlorite	Merck	Cat# XX0637
Sodium thiosulfate	Merck	Cat# 106512
PLURONIC F-127	Merck	Cat# P2443-250G
Albumin from bovine serum	Sigma-Aldrich	Cat# A7030
HiTrap Heparin HP 1 mL column	Merck	Cat# GE17-0407-01
Streptavidin-coated polystyrene particles 0.5% w/v	Spherotech	Cat# SVP-40-5
Ni-NTA agarose resin	Qiagen	Cat# 30210
Lambda DNA	Thermo Fisher	Cat# SD0011
Recombinant X. laevis histones	The Histone Source, Protein Expression and Purification Facility, Colorado State University	N/A
RAD51	This study	N/A
RAD51(A647)	This study	N/A
hRPA	This study	N/A
BRCA2	This study	N/A
BRCA2-eGFP	This study	N/A
BRCA2-N	This study	N/A
BRCA2-C	This study	N/A
BRCA2-C2	This study	N/A
T4 Polynucleotide Kinase	NEB	Cat#M0201S
T4 DNA Ligase	NEB	Cat# M0202S
T4 DNA Ligase Reaction Buffer	NEB	Cat# B0202S
SYTOX Orange Nucleic Acid Stain	Thermo Fisher	Cat#S11368
S. p. Cas9 D10A nickase	IDT	Cat# 1081062
EDTA-free cOmplete protease inhibitor cocktail	Roche	Cat# COEDTAF-RO
PhosSTOP phosphatase inhibitor cocktail	Roche	Cat# PHOSS-RO
4x NuPAGE LDS sample buffer	Themo Fisher	Cat# NP0008
Clarity Western ECL	Bio-Rad	Cat# 1705061
Clarity Max Western ECL	Bio-Rad	Cat# 1705062
Critical commercial assays
QIAquick PCR Purification Kit	Qiagen	Cat# 28104
Q5 Site-Directed Mutagenesis Kit	New England BioLabs	Cat# E0554
Deposited data
Original images with cropped areas marked	Mendeley	Mendeley: https://doi.org/10.17632/sgw9t49xyg.1
Original code for kymograph/image analysis	Zenodo	Zenodo: https://doi.org/10.5281/zenodo.7857347
Experimental models
Spodoptera frugiperda (Sf9) insect cells	Crick Cell Services	N/A
Trichoplusia ni High Five (BTI-TN-5B1-4) insect cells	Thermo Fisher	Cat# B85502
Oligonucleotides
Oligonucleotides are listed in Table S1	IDT and Sigma Aldrich	Table S1
Recombinant DNA
pFBDM-BRCA2-N	This study	N/A
pFBDM-BRCA2-C	This study	N/A
pFBDM-BRCA2-C2	This study	N/A
pFB-BRCA2	This study	N/A
pFB-BRCA2-eGFP	This study	N/A
pBIG1a-hRFA1/hRFA2/hRFA3-his10	This study	N/A
pET11c-RAD51	Dr. Lumir Krejci	N/A
pET11c-RAD51 C319S	This study	N/A
pFastBac1-hRFA1	This study	N/A
pFastBac1-hRFA2	This study	N/A
pFastBac1-hRFA3-his10	This study	N/A
Software and algorithms
GraphPad Prism 9	Graphpad	https://www.graphpad.com/scientific-software/prism/
Fiji	Open source	https://imagej.net/Fiji
Matlab R2018b (9.5.0)	MathWorks	https://uk.mathworks.com
Lumicks Pylake	Python package from Lumicks	https://lumicks-pylake.readthedocs.io/en/latest/index.html#
Other
C-trap optical trapping and confocal microscopy setup	Lumicks	N/A