The role of chromatin in the catalysis of homologous strand pairing by Rad54 and Rad51 was investigated. Rad54 is related to the ATPase subunits of chromatin-remodeling factors, whereas Rad51 is related to bacterial RecA. In the absence of superhelical tension, the efficiency of strand pairing with chromatin is >100-fold higher than that with naked DNA. In addition, 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 (Alexiadis, 2002).
Homologous recombination occurs in the repair of DNA double-strand breaks as well as during meiosis. Genetic studies in Saccharomyces cerevisiae led to the identification of the RAD52 epistasis group of genes (which includes RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, MRE11, and XRS2) as components of the recombinational repair pathway. These genes are conserved from yeast to humans. A central protein in this pathway is Rad51, which is related to the bacterial RecA protein. Both Rad51 and RecA are able to mediate strand invasion and annealing to yield a D loop, which is a key step in the recombination process. In this reaction, Rad51 (or RecA) forms a nucleoprotein filament on single-stranded DNA in the presence of ATP, and this filament is used for homologous pairing with a double-stranded DNA molecule. The efficiency of strand pairing by Rad51 (between single-stranded DNA and homologous duplex DNA) has been shown to be stimulated by the presence of additional factors such as RP-A, the Rad55-Rad57 heterodimer, Rad52, and Rad54 (Alexiadis, 2002 and references therein).
To study homologous recombination in the context of chromatin, focus was placed on the ability of purified recombinant Rad51 and Rad54 to catalyze D-loop formation between single-stranded DNA and homologous double-stranded DNA that is packaged into chromatin. The function of Rad54 in chromatin is of particular interest because it is a member of the Snf2-like family of ATPases. The Snf2-like family includes proteins such as Swi2/Snf2, Sth1, ISWI, Ino80, and Mi-2/CHD3/CHD4, which are the ATPase subunits of chromatin-remodeling factors that catalyze the mobilization of nucleosomes. It thus seemed possible that Rad54 would be important for homologous recombination in chromatin. Therefore, whether purified Rad51 and Rad54 can mediate D-loop formation with chromatin was investigated (Alexiadis, 2002).
To study the biochemical properties of Rad51 and Rad54, focus was placed Drosophila Rad51 and Rad54 (with C-terminal Flag tags) in Sf9 cells by using a baculovirus expression system, and then the proteins were purified to near homogeneity by FLAG immunoaffinity chromatography. The ability of these factors to mediate D-loop formation between a radiolabeled, single-stranded oligonucleotide (termed DL2; 135 nt) and a homologous, double-stranded plasmid (pU6LNS; 3291 bp) was tested. In this reaction, Rad51 assembles onto the single-stranded oligonucleotide in the presence of ATP to give a nucleoprotein filament, and then Rad54 interacts with the Rad51-oligonucleotide complex and facilitates the strand-pairing reaction. These experiments reveal that purified recombinant Drosophila Rad51 and Rad54 can catalyze the formation of D loops in a manner that is dependent on Rad51, Rad54, ATP, and homologous plasmid DNA (Alexiadis, 2002).
Next, the ability of Rad51 and Rad54 to catalyze D-loop formation in chromatin was tested. In these experiments, chromatin was reconstituted by salt dialysis techniques. The salt dialysis chromatin (SD chromatin) was prepared by gradually decreasing the salt concentration in a mixture of plasmid DNA and purified core histones from Drosophila embryos, and fully reconstituted chromatin was separated from partially reconstituted chromatin by sucrose gradient sedimentation. Micrococcal nuclease digestion analysis of the chromatin samples revealed that the salt dialysis chromatin consisted of closely packed arrays of nucleosomes. D-loop reactions were performed with the SD chromatin. These experiments revealed that Rad51 and Rad54 are able to form D loops with SD chromatin at an efficiency that is slightly higher than that obtained with naked DNA. Moreover, the rate of D-loop formation by Rad51 and Rad54 with chromatin is similar to that seen with naked DNA. In contrast, the Escherichia coli RecA protein is able to mediate D-loop formation with naked DNA, but not with chromatin. Thus, these experiments, which were performed with completely purified components, show that Rad51 in cooperation with Rad54 can mediate D-loop formation with chromatin with comparable efficiency and kinetics as with DNA, whereas the bacterial recombinase RecA is unable to mediate strand pairing with chromatin. The inability of RecA to function with chromatin is consistent with previous studies carried out with mononucleosomes, and further suggests that RecA is lacking a chromatin-specific function that is present in Rad51 and/or Rad54. In this regard, whether Rad54 could stimulate D-loop formation in chromatin by RecA was tested, but no activity was demonstrated (Alexiadis, 2002).
The bulk of the eukaryotic genome appears to possess little superhelical tension, and therefore the effect of torsional stress upon D-loop formation by Rad51 and Rad54 was investigated. To this end, the salt dialysis chromatin was relaxed with purified topoisomerase I. The salt dialysis chromatin was reconstituted by using supercoiled plasmid DNA in the absence of topoisomerases. Under these conditions, the DNA remains chemically unchanged, since no phosphodiester bonds are broken. Hence, in the absence of topoisomerase I, the numbers of supercoils in the naked DNA and chromatin (which was deproteinized prior to electrophoresis) are essentially identical. When topoisomerase I is added to the chromatin, the unconstrained supercoils are relaxed, but upon deproteinization, the resulting DNA exhibits supercoils that are caused by the wrapping of the DNA in nucleosomes, because the wrapping of the DNA around each histone octamer constrains approximately one negative supercoil (Alexiadis, 2002).
Strand-pairing reactions were performed with DNA and salt dialysis chromatin in the absence or presence of topoisomerase I. With naked DNA, a >100-fold reduction in the efficiency of D-loop formation was observed upon relaxation of the template with topoisomerase I. Notably, this >100-fold decrease in strand-pairing efficiency is much more pronounced than the twofold reduction seen with yeast Rad51, Rad54, and RPA. This difference could potentially be due to the use of yeast versus Drosophila factors, the presence or absence of RPA, the length of the single-stranded DNA (5386 nt, Van Komen, 2000; 135 nt, this study), and/or the concentration of Rad51 in the reaction medium (1500 nM, Van Komen, 2000; 200 nM, this study). Note, however, that stimulation of D-loop formation by purified RPA was not observed in reactions performed in this study. In contrast to the effects seen with naked DNA, relaxation of the chromatin by topoisomerase I has little effect on the efficiency of D-loop formation by Rad51 and Rad54. Thus, in the absence of superhelical tension, strand pairing by Rad51 and Rad54 occurs with higher efficiency in chromatin than in naked DNA (Alexiadis, 2002).
Because Rad54 is related to the ATPase subunit of chromatin-remodeling complexes, whether Rad54 possesses chromatin-remodeling activity was tested. The ability of Rad54 and/or Rad51 to facilitate the access of a restriction enzyme (HaeIII) to DNA packaged into nucleosome arrays was tested. ACF was used as a positive control. This type of restriction-enzyme accessibility assay has been used for the analysis of chromatin remodeling in vivo, the biochemical purification of the CHRAC chromatin-remodeling factor, the characterization of the INO80.com remodeling complex, and the comparative analysis of six chromatin-remodeling complexes (ySWI/SNF, yRSC, hSWI/SNF, xMi-2, dCHRAC, dNURF). Neither Rad54 alone nor Rad51 alone exhibits any detectable chromatin-remodeling activity in the absence or presence of the DL2 oligonucleotide. In sharp contrast, Rad54 and Rad51 function cooperatively in the ATP-dependent remodeling of chromatin. The ability of Rad54 and Rad51 to rearrange chromatin structure is consistent with their ability to catalyze strand pairing with chromatin. It is also notable that Rad54 requires the presence of Rad51 to function as a chromatin-remodeling factor (Alexiadis, 2002).
In conclusion, these studies have revealed that D-loop formation by Rad54 and Rad51 occurs with >100-fold higher efficiency with chromatin relative to naked DNA in the absence of superhelical torsion. In addition, Rad54 and Rad51 act cooperatively in the ATP-dependent remodeling of chromatin. This ability of Rad54 and Rad51 to alter chromatin structure is likely to be related to their chromatin-specific function in the strand-pairing reaction. These findings provide an example of optimized function of eukaryotic DNA-using proteins in chromatin. Moreover, it is possible that the use of chromatin templates, instead of naked DNA templates, might similarly increase the efficiency of targeted homologous recombination in vivo (Alexiadis, 2002).
Biochemical studies of the BLM gene product have shown its ability in conjunction with topoisomerase IIIalpha to resolve double Holliday structures through a process called 'dissolution.' This process could prevent crossing over during repair of double-strand breaks. This study describes an analysis of the Drosophila BLM gene, DmBlm, in the repair of double-strand breaks in the premeiotic germ line of Drosophila males. With a repair reporter construct, Rr3, and other genetic tools, it is shown that DmBlm mutants are defective for homologous repair but show a compensating increase in single-strand annealing. Increases of 40- to 50-fold in crossing over and flanking deletions also were seen. Perhaps most significantly, the template used for homologous repair in DmBlm mutants is itself subject to deletions and complex rearrangements. These template disruptions are indicative of failure to resolve double Holliday junctions. These findings, along with the demonstration that a weak allele of topoisomerase IIIalpha has some of the same defects as DmBlm, support the dissolution model. Finally, an analysis of DmBlm mutants in conjunction with mus81 or spnA (Rad51) reveals a second function of BLM distinct from the repair of induced double-strand breaks and possibly related to maintenance of replication forks (Johnson-schlitz, 2006).
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