Choosing a strategy for reconstitution of DSBs repair in vitro
For reconstitution we used purified human proteins: Rad51, Rad52, Rad54, RPA, and DNA polymerase η4-8. Rad51 was found to promote formation of joint molecules (D-loops) (Fig. 1a)4. RPA, a ubiquitous ssDNA-binding protein, was included because of its important role in DSB repair in vivo, where it interacts with ssDNA-intermediates and DNA repair proteins9. DNA polymerase η was used for extension of the invading DNA because recent data implicated it in recombinational repair of DSB (Fig. 1b)10. Rad54 protein was used for dissociation of extended joint molecules, in accord with our recent data (Fig. 1c)3. Rad52 was expected to promote annealing of the tailed DNA displaced from the D-loop with the complementary tailed DNA, which represented the second end of broken DNA (Fig. 1d)11. This set of proteins defines the minimal DSBs repair reconstitution system.
Design of DNA substrates for DSB repair reconstitution system
1| In the DBS reconstitution system tailed DNAs (I and II) represent two parts of broken chromosome processed by specific exonucleases (Fig. 2a). Both substrates were prepared by annealing of 100-mer oligonucleotides with 36-mer oligonucleotides to generate tailed DNA structures. Before annealing, one of the 100-mer oligonucleotides (a component of tailed DNA I) was labeled using [γ-32P]ATP and T4 polynucleotide kinase, followed by DNA purification through the Micro-BioSpin 6 column (Bio-Rad).
2| Single-stranded part of tailed DNA (I) was designed to be complementary to the region of supercoiled (sc) pUC19 dsDNA, which represented homologous undamaged chromosome used as a template in DSB repair by HR. The 3’-end of tailed DNA (I) was expected to invade the scDNA forming D-loop (Fig. 2b).
3| After D-loop formation, DNA polymerases may use the 3’-end of the invading strand as a primer to restore DNA sequences lost during DSB formation (Fig. 2c). To control the length of the primer extension the target region of plasmid DNA was chosen in a such way that only a limited 32 nt DNA synthesis was allowed in the presence of three dNTPs: dATP, dCTP, and dTTP (Fig. 2c).
4| After D-loop dissociation, the extended region of tailed DNA (I) can anneal to tailed DNA (II) (Fig. 2d). This annealing would generate a DNA product resembling the product of DSB repair by HR.
5| The remaining steps of DSB repair (not covered by this protocol) include filling of DNA gaps by DNA polymerases and sealing DNA nicks by DNA ligases.
Reconstitution of DSB repair in vitro
1| The initial mixture was of the following composition: 25 mM Tris acetate, pH 7.5, 1 mM ATP, 2 mM magnesium acetate, 2 mM calcium chloride, 2 mM DTT, BSA (100 μg/ml), 20 mM phosphocreatine, creatine phosphokinase (30 units/ml), dNTPs (dATP, dTTP, dCTP; 100 μM each), DNA polymerase η (1.5 ng/μl), and 32P-labeled tailed DNA (I) (30 nM, molecules). All incubations were performed at 37 °C. The reactions were stopped by addition of proteinase K (800 μg/ml) and 1.2% SDS. To prevent spontaneous annealing of the product of extension and the tailed DNA (II) during deproteinization, a 32-mer oligonucleotide (1.2 μM, molecules) that is complementary to tailed DNA (II) was added with the stop buffer. The DNA products of D-loop dissociation were deproteinized and analyzed by electrophoresis in an 8% polyacrylamide gel. If desired, the products could be also analyzed in a 1 % agarose gel, in parallel. The DNA products were visualized and quantified using a Storm 840 PhosphorImager (GE Healthcare).
2| The reconstitution was performed by adding all proteins and DNA substrates in three steps in the indicated order followed by incubation for the indicated periods of time (Fig. 3a): RPA (225 nM) for 5 min, Rad51 (1 μM) for 15 min, pUC19 scDNA (50 μM, nucleotides) for 15 min, 2 mM of EGTA and tailed DNA (II) (30 nM, molecules) for 5 min, and finally Rad54 (200 nM) and Rad52 (1.5 μM) followed by 30 min incubation. In controls, by omitting individual protein and DNA constituents we analyzed their effects on DSB repair.
3| In the first step, 3’-tailed DNA (I) representing an early intermediate of DSB repair was mixed with DNA polymerase η and RPA. Then Rad51 protein was added to form a filament with tailed DNA. In the second step, D-loop formation was initiated by addition of pUC19 scDNA; omission of Rad51 prevented D-loop formation (Fig. 3b, lane 1). Using the 3’-end of the invading DNA as a primer, DNA polymerase η commenced DNA synthesis. The extended DNA could be visualized in a polyacrylamide gel after D-loop dissociation with Rad54 (Fig. 3b, lanes 5, 7-9). In the third step, the tailed DNA (II) complementary to the extended segment of the tailed DNA (I) was added together with 2 mM EGTA that chelated Ca2+ followed by addition of Rad54 and Rad52 proteins.
4| Rad54 catalyzed dissociation of both the original D-loops, when DNA polymerase η was omitted (Fig. 3b, lane 3) and D-loops extended with DNA polymerase η (Fig. 3b, lanes 5, 7-9). Omission of Rad54 left the D-loops intact (Fig. 3b, lanes 2, 4 and 6).
5| Rad52 annealed the extended tailed DNA (I) dissociated from the D-loops with the complementary tailed DNA (II) (Fig. 3b, lane 9); Rad52 omission prevented annealing (Fig. 3b, lane 8). Omission of RPA rendered Rad52 dispensable, since after D-loops dissociation with Rad54 tailed DNAs could anneal either spontaneously or with an assistance of Rad51 or Rad54 proteins (Fig. 3b, lane 7). Overall, the reactions yielded an expected DSB repair product directly demonstrating the feasibility of the SDSA mechanism of DSB repair.