Tailed duplex DNA is the preferred substrate for Rad51 protein-mediated homologous pairing - PubMed (original) (raw)

Tailed duplex DNA is the preferred substrate for Rad51 protein-mediated homologous pairing

A V Mazin et al. EMBO J. 2000.

Abstract

The repair of potentially lethal DNA double-stranded breaks (DSBs) by homologous recombination requires processing of the broken DNA into a resected DNA duplex with a protruding 3'-single-stranded DNA (ssDNA) tail. Accordingly, the canonical models for DSB repair require invasion of an intact homologous DNA template by the 3'-end of the ssDNA, a characteristic that the bacterial pairing protein RecA possesses. Unexpectedly, we find that for the eukaryotic homolog, Rad51 protein, the 5'-end of ssDNA is more invasive than the 3'-end. This pairing bias is unaffected by Rad52, Rad54 or Rad55-57 proteins. However, further investigation reveals that, in contrast to RecA protein, the preferred DNA substrate for Rad51 protein is not ssDNA but rather dsDNA with ssDNA tails. This important distinction permits the Rad51 proteins to promote DNA strand invasion using either 3'- or 5'-ends with similar efficiency.

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Figures

Fig. 1. The pairing bias exhibited by the eukaryotic Rad51 proteins is opposite to that of prokaryotic RecA protein. (A) Scheme of the experiments. Rad51 and RecA nucleoprotein filaments were formed on one of two complementary ssDNA substrates (63mers, #1 or 2). DNA strand exchange was initiated by addition of 32P-labeled dsDNA (31mers, #45 and 55) (12 μM) that was homologous to either the 5′- or 3′–terminal regions of oligonucleotides #1 and 2, respectively. The straight line indicates homology to the dsDNA, the zig-zag line indicates heterology; asterisks denote the 32P-labeled strand of dsDNA. (B, C and D) The kinetics of DNA strand exchange promoted by yeast Rad51 protein, human Rad51 protein and E.coli RecA protein, respectively.

Fig. 2. In Rad51 protein-mediated invasion of supercoiled DNA, linear ssDNA with homology at the 5′–end is more invasive than ssDNA with homology at the 3′–end. (A) Scheme of the experiments. (B) The kinetics of joint molecule formation promoted by Rad51 protein between supercoiled pUC19 dsDNA and partially homologous ssDNA oligonucleotides SK#3 and SK#5, carrying homology at either the 3′- or 5′–terminal region, respectively. (C) Identical reactions to those in (B), except that they were supplemented with Rad54 protein (0.15 μM). (D) Reactions promoted by RecA protein with the same DNA substrates. The percentage of joint molecule products was expressed relative to the limiting amount of plasmid DNA.

Fig. 3. Duplex DNA with an ssDNA tail is the preferred substrate for DNA strand exchange promoted by Rad51 protein. (A) The kinetics of DNA strand exchange promoted by Rad51 protein. Rad51 nucleoprotein filaments were formed on either ssDNA (63mer, #2 or 31mer, #55) or tailed dsDNA with a 3′ single-stranded end (63mer, #2 and 32mer, #5). DNA strand exchange was initiated by addition of 32P-labeled dsDNA (31mers, #45 and 55) that was homologous to either the 3′–terminal region of both the 63mer ssDNA and 32/63mer tailed dsDNA or to the entire 31mer ssDNA. (B) The kinetics of DNA strand exchange promoted by RecA protein. RecA nucleoprotein filaments were formed on either ssDNA (63mer, #1) or tailed dsDNA with a 5′ single-stranded end (63mer, #1 and 32mer, #6). DNA strand exchange reactions were initiated by addition of 32P-labeled dsDNA (31mers, #45 and 55). In order to measure accurately the initial rates, the reactions with RecA protein were carried out at 24°C. The drawings above each panel illustrate the DNA strand exchange reactions promoted by each protein. The straight lines indicate homology to the dsDNA, the zig-zag lines indicate heterology; asterisks denote the 32P-labeled strand of dsDNA.

Fig. 4. Both yeast and human Rad51 proteins show a preference for tailed duplex DNA in DNA strand exchange. (A and B) The kinetics of DNA strand exchange promoted by yeast or human Rad51 protein. Rad51 nucleoprotein filaments were formed on either ssDNA (63mers, #1 or 2) or tailed dsDNA with a 5′ single-stranded end (63mer, #1 and 32mer, #6) or a 3′ single-stranded end (63mer, #2 and 32mer, #5). DNA strand exchange reactions were initiated by addition of 32P-labeled dsDNA (31mers, #45 and 55) that was homologous to either the 3′- or 5′–terminal regions of the ssDNA or the tailed dsDNA; the straight line indicates homology to the dsDNA, the zig-zag line indicates heterology. Since only one strand of dsDNA was labeled, DNA strand exchange with the 3′ or 5′ homologous ssDNA substrates produces displaced 32P-labeled 31mer ssDNA or 32P-labeled 32/63mer heteroduplex, respectively (see Figure 1A). In (C), the initial rates of DNA strand exchange were calculated for the first minute of the reactions and were normalized relative to the rate obtained for the 5′–tailed dsDNA (100%) for each protein. Placement of the 32P-label in the opposite dsDNA strand had no effect on the kinetics of joint molecule formation.

Fig. 5. A stoichiometric concentration of Rad51 protein is required for optimal DNA strand exchange activity with either ssDNA or tailed dsDNA substrates. Nucleoprotein filaments were formed by adding yeast Rad51 protein at the indicated concentrations to either ssDNA (63mer, #2) (12 μM) or tailed dsDNA with a 3′ single-stranded end (63mer, #2 and 32mer, #5) (18 μM) in the presence of 2 mM ATP or 1 mM ATPγS. DNA strand exchange was initiated by addition of 32P-labeled dsDNA (31mers, #45 and 55) that was homologous to the 3′–terminal region of the ssDNA or the tailed dsDNA substrate. The reactions were carried out for 5 min. RPA was omitted in both reactions. Reactions were carried out in the presence of ATP (A) and ATPγS (B).

Fig. 6. RPA inhibits DNA strand exchange with tailed dsDNA less than with ssDNA substrates. (A) The kinetics of DNA strand exchange promoted by yeast Rad51 protein when RPA was pre-bound to either ssDNA or 3′–tailed dsDNA. Reactions were identical to those described in the legend to Figure 1, except that RPA (3 μM) was added to either ssDNA (63mer, #2 or 31mer, #55) or 3′–tailed dsDNA (63mer, #2 and 32mer, #5), and was followed by a 7 min incubation prior to addition of yeast Rad51 protein. Homologous dsDNA was added 3 min after addition of Rad51 protein. For comparison, (B) shows the kinetics of identical DNA strand exchange reactions except that RPA was omitted.

Fig. 7. Rad51 protein displays a hierarchy of complex formation with different DNA substrates. DNA substrates, dsDNA 63mer (63mers, #1 and 2) (24 μM), dsDNA 31mer (31mers, #45 and 55) (24 μM), tailed dsDNA 32/63mers (63mer, #2 and 32mer, #5) (18 μM) and ssDNA 63mer (63mer, #1) (12 μM), were mixed with increasing concentrations of yeast Rad51 protein and analyzed. (A) shows images of the gels; (B) represents quantitation of the PhosphorImager scans.

Fig. 8. Rad51 protein binds preferentially to duplex DNA with ssDNA tails. DNA substrates, dsDNA (63mers, #1 and 2) (24 μM) or tailed dsDNA (63mer, #2 and 32mer, #5) (18 μM) (both 32P-labeled), were mixed with various concentrations of unlabeled dsDNA (63mers, #41 and 49). These mixtures were incubated for 15 min with yeast Rad51 protein (7.8 μM) in the presence of ATP. Rad51 protein–DNA complexes were analyzed by gel electrophoresis and quantified. The inset shows the data from an identical experiment, except that it was performed in the presence of ATPγS instead of ATP.

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Tailed duplex DNA is the preferred substrate for Rad51 protein-mediated homologous pairing - PubMed (original) (raw)