DNA with Different Local Torsional States Affects RecA‐Mediated Recombination Progression

Abstract

DNA topology is thought to affect DNA enzyme activity. The helical structure of duplex DNA dictates the change of topological states during strand separation when DNA is constrained. During the repair of DNA double-stranded breaks, the RecA nucleoprotein filament invades DNA and carries out consecutive strand exchange reactions coupled with duplex DNA strand separation. It has been suggested that torsional strain could be generated and its accumulation could inhibit strand exchange. We used hairpin and nicked DNA substrates to test how torsional strain alters the RecA-mediated strand exchange efficiency. Single-molecule tethered particle motion (TPM) experiments showed that torsionally constrained hairpin DNA substrates returned nearly no successful strand exchange events catalyzed by RecA. Surprisingly, the strand exchange efficiencies increase in the presence of DNA nicks or loop disruption. The dwell time of transient RecA events in hairpin is shorter compared to those found in nicked or fork DNA substrates, which suggests a limited strand exchange progression in hairpin substrates. Our observation shows that RecA generates local torsional strain during strand exchange, and the inability to dissipate this torsional strain inhibits homologous recombination progression. DNA topological states are thus important regulation measures of DNA recombination.

A 5′-to-3′ strand exchange polarity is intrinsic to RecA nucleoprotein filaments in the absence of ATP hydrolysis

“…In this invading strand experiment, a hybrid DNA substrate containing ssDNA overhangs (40 nt) on both ends is prepared by annealing a long (229 nt) and a short (149 nt) single-stranded DNA and is anchored on an anti-digoxigenin-coated coverglass (Figure 1A). The inclusion of the single-stranded region in the hybrid DNA efficiently increases invading events of the initiation of strand exchange (50,51). A comparable single-stranded region is present at both ends of the duplex so as not to bias initiation at one end or the other.…”

“…Escherichia coli wild-type RecA protein and RecAΔC17 were purified as described (49). Hybrid DNA substrates were generated using the protocol described earlier (50,51), with all the primers listed in Supplementary Table SI. The short homologous hybrid DNA substrate used here is a 229/149 nt DNA, with a 40 nt overhang at both its 5′- and 3′-proximal ends.…”

The Role of Topoisomerase II in DNA Repair and Recombination in Arabidopsis thaliana

“…When the DNA double helix is negatively supercoiled, the ssDNA filament loaded with RecA (the bacterial equivalent to RAD51/DMC1) is more efficient in invading and forming a joint molecule [ 69 , 70 ]. Lu and Li [ 23 ] proposed the participation of topoisomerases in releasing this stress, because DNA substrates with a nick allow RecA strand exchange four times more efficiently than in the absence of a nick. In fact, topoisomerase I has been shown to physically bind RecA and help the homology search [ 71 ].…”

“…Biochemical evidence indicates HJ formation requires negative supercoiling in the centre of this four-way DNA junction, and to compensate this tension, positive supercoiling is generated at both adjacent sides of the molecule [ 22 ]. Furthermore, stable strand invasion has also been reported to required negative supercoiling in the template strand and topoisomerases have been proposed to help achieve this conformation [ 23 ]. Evidence of a role for TOPII in HR mediated DSB repair was reported by Morotomi-Yano and colleagues [ 24 ].…”

Quantitation of DNA-Binding Affinity Using Tethered Particle Motion

“…Although the L p cannot be directly calculated with TPM, the change in RMS is a reliable measure for protein binding. TPM has been used to measure DNA-binding by architectural proteins, proteins involved in DNA replication, transcription, DNA repair, and recombination [12][13][14][15][16][17][18][19][20][21]. A bead is tethered to a glass surface by DNA.…”