Strand pairing by Rad54 and Rad51 is enhanced by chromatin

Abstract

We investigated the role of chromatin in the catalysis of homologous strand pairing by Rad54 and Rad51. Rad54 is related to the ATPase subunits of chromatin-remodeling factors, whereas Rad51 is related to bacterial RecA. In the absence of superhelical tension, we found that the efficiency of strand pairing with chromatin is >100-fold higher than that with naked DNA. In addition, we observed that Rad54 and Rad51 function cooperatively in the ATP-dependent remodeling of chromatin. These findings indicate that Rad54 and Rad51 have evolved to function with chromatin, the natural substrate, rather than with naked DNA.

Figure 1

Drosophila Rad51 and Rad54 mediate D-loop formation. (A) Synthesis and purification of Drosophila Rad51 and Rad54. Flag-tagged Drosophila Rad51 and Rad54 were synthesized in Sf9 cells by using a baculovirus expression vector and affinity-purified with monoclonal antibodies that recognize the Flag epitope. The proteins were subjected to 10% polyacrylamide–SDS gel electrophoresis. The proteins were visualized by staining with Coomassie Brilliant Blue R-250. (B) Formation of D loops with purified Drosophila Rad51 and Rad54 proteins. In the Complete reaction, Rad51 was preincubated with radiolabeled DL2 oligonucleotide in the presence of ATP at 27°C for 20 min; Rad54 and a homologous supercoiled plasmid DNA (pU6LNS) were added; and then the reaction was allowed to proceed at 27°C for 4 min. The resulting DNA was deproteinized, and the samples were subjected to agarose gel electrophoresis and autoradiography. Other reactions either were missing a single component, as indicated, or contained an equivalent mass of nonhomologous DNA. The final concentrations of the reaction components were as follows: Rad51, 200 nM; Rad54, 46 nM; ATP, 2 mM; DL2 oligonucleotide, 1 nM; and pU6LNS, 4 nM.

Figure 2

Rad51 and Rad54, but not RecA, are able to mediate D-loop formation with chromatin. (A) Micrococcal nuclease digestion analysis of chromatin reconstituted from purified components by salt dialysis. Purified Drosophila core histones were reconstituted into chromatin by using salt dialysis techniques (Jeong et al. 1991). The samples were subjected to partial digestion with two different concentrations of micrococcal nuclease and subsequently deproteinized. The resulting DNA fragments were resolved by agarose gel electrophoresis and visualized by staining with ethidium bromide. The mass markers (M) are the 123-bp DNA ladder (GIBCO-BRL). (B) Comparison of the ability of Rad51 + Rad54 versus RecA to mediate D-loop formation with either naked DNA or salt dialysis chromatin. Reactions with Rad51 and Rad54 were performed as in Figure 1B with naked DNA or salt dialysis chromatin (SD Chromatin), except that the final concentration of Rad54 was 28 nM and that of DNA or chromatin was 1 nM. Reactions with RecA were performed in an analogous manner by incubation of purified Escherichia coli RecA with radiolabeled DL2 oligonucleotide at 27°C for 20 min, followed by the addition of plasmid DNA and incubation at 27°C for an additional 20 min. The final concentration of RecA protein was 870 nM. (C) Kinetics of D-loop formation with naked DNA and chromatin. Reactions were performed as in B, except that they were allowed to proceed for the indicated times after the addition of Rad54 and homologous DNA.

Figure 3

Chromatin enhances D-loop formation by Rad51 and Rad54 in the absence of superhelical tension. (A) Relaxation of DNA and chromatin by topoisomerase I. Plasmid DNA and chromatin (reconstituted by salt dialysis) were relaxed with purified recombinant Drosophila topoisomerase I (catalytic fragment). An aliquot of each of the samples was deproteinized and subjected to 1% agarose gel electrophoresis in the presence of 5 μM chloroquine followed by staining with ethidium bromide. ++ indicates twice the topoisomerase I concentration as that used in the + lanes. (B) D-loop formation with relaxed chromatin. D-loop reactions were performed as in Figure 1B, with equimolar amounts of the DNA and chromatin samples shown in A. The topoisomerase I remained in the samples throughout the strand-pairing reactions.

Figure 4

The packaging of relaxed DNA into chromatin facilitates strand pairing by Rad51 and Rad54. (A) ACF-mediated chromatin assembly. Chromatin assembly reactions were performed with purified ACF, NAP-1, topoisomerase I, plasmid DNA, and ATP in the presence or absence of purified core histones, as indicated. The reaction products were subjected to micrococcal nuclease digestion analysis. (B) Strand-pairing reactions. The samples in A were used in strand-pairing reactions with purified Drosophila Rad51 and Rad54 along with the DL2 oligonucleotide. D-loop reactions were performed as in Figure 3B, except that the final concentration of Rad54 was 27 nM. The effect of nonnucleosomal histones on strand pairing was also tested by the addition of core histones (the same amount as that used in the center lane) to the DNA after mock chromatin assembly (with ACF, NAP-1, DNA, ATP, and topoisomerase I in the absence of core histones) and immediately prior to the strand-pairing reactions (right lane).

Figure 5

Rad54 and Rad51 function cooperatively in the remodeling of chromatin. Restriction enzyme accessibility assays were carried out with naked DNA or chromatin (salt dialysis reconstitution), the indicated factors, and the restriction enzyme HaeIII (15 units, GIBCO-BRL) in the same reaction medium used for D-loop reactions. The reactions were incubated at 27°C for 1 h. The samples were deproteinized and subjected to electrophoresis on a 1% agarose gel. The DNA was visualized by staining with ethidium bromide. The final concentrations of the components, which were included as indicated, were as follows: plasmid DNA or chromatin, 2 nM; DL2 oligonucleotide, 1 nM; ATP, 2 mM; Rad51, 200 nM; Rad54, 46 nM; and ACF, 3 nM. The amount of remodeling observed increases with the concentration of the factors (Rad51 and Rad54) as well as with the reaction time (data not shown). Reactions containing DL2 oligonucleotide and Rad51 were preincubated at 27°C for 20 min. There are 14 HaeIII sites in the pU6LNS plasmid, one of which is in the homologous pairing site.