(Received for publication, August 10, 1995; and in revised form, October 23, 1995)
From the
Processing of DNA damage by the DNA double-strand break repair pathway in mammalian cells is accomplished by multiprotein complexes. However, the nature of these complexes and details of the molecular interactions are not fully understood. Interaction of the yeast RAD51 and RAD52 proteins plays a crucial role in yeast DNA homologous recombination and DNA double-strand break repair. Here, specific interactions between human RAD51 and RAD52 proteins are demonstrated both in vivo, using the yeast two-hybrid system and immunoprecipitation of insect cells co-infected with RAD51 and RAD52 recombinant viruses, and in vitro, using affinity chromatography with purified recombinant proteins. These results suggest that RAD52 may modulate the catalytic activities of RAD51 protein such as homologous pairing and strand exchange through a direct physical interaction. In addition, the domain in RAD52 that mediates this interaction was determined in vitro and in vivo. The RAD51-interacting region (amino acids 291-330) of the human RAD52 protein shows no homology with the yeast RAD52 protein, indicating that the interaction between RAD51 and RAD52 is species-specific.
The genome of living organisms is constantly damaged by exposure
to exogenous and endogenous agents. The inherent stability of the DNA
molecule provides the first level of protection from such damage.
Moreover, organisms have developed intricate machineries of DNA repair
to fix various types of damage. During the last 2 decades,
investigators have identified and characterized different DNA repair
pathways using various approaches such as yeast genetic studies. Three
epistasis groups have been identified in Saccharomyces cerevisiae that control nucleotide excision repair (RAD3 epistasis group),
error prone/post-replication repair (RAD6 epistasis group), and
recombinational repair (RAD52 epistasis group). Both genetic and
molecular studies on the yeast RAD52 epistasis group have clearly
indicated close links between the mechanism of homologous recombination
and DNA double-strand break (DSB) ()repair. Mutants
belonging to this group exhibit elevated sensitivity to ionizing
radiation and methyl methanesulfonate. In addition, this group of
mutants is defective in both meiotic and mitotic recombination
processes (1, 2, 3, 4, 5) .
Most biochemical processes in various mammalian DNA repair require multiprotein complexes. Efficient removal of photodamages or bulky adducts requires more than a dozen proteins in the nucleotide excision repair(6, 7) . This is also the case for mismatch repair machineries (8) . Recently, a mammalian protein complex that repairs double-strand breaks by recombination has been identified(9) . Genetic and physical evidence for the interaction of yeast RAD52 and RAD51 suggests that at least a subset of the RAD52 epistasis group proteins interact with each other(10, 11, 12) . Additional evidence for a multiprotein repair complex comes from the dominant negative phenotypes exhibited by mutant alleles of RAD52. Some rad52 dominant-negative alleles act via nonproductive interaction with RAD51, whereas others can act independently of RAD51, perhaps associating with other repair proteins. Therefore, RAD52 epistasis group-mediated DSB repair appears to require multiprotein complexes for damage processing.
The null mutant of RAD51 is partially defective in the formation of physical recombinants and accumulates double-strand breaks at a meiotic recombination hot spot(10, 13) . RAD51 from yeast and other eukaryotes has considerable homology to bacterial RecA(10, 14, 15, 16, 17, 18, 19, 20, 21) , which has been shown to play a major role in recombination-mediated DNA repair in bacteria. RecA promotes homologous pairing and DNA strand exchange by forming nucleofilaments in the presence of ATP(22, 23, 24) . Recently, yeast RAD51 has been shown to form nucleofilaments (25) and possess biochemical characteristics similar to RecA(26) . RAD51 can catalyze homologous pairing and strand exchange activities in vitro(26) . The human RAD51 protein has also been shown to form nucleofilaments with DNA(27) , and forms nuclear foci after ionizing radiation(28) .
Most of the information regarding the functions of the RAD52 protein has come from studies in yeast. Some studies have suggested that the RAD52 protein is not required for the initiation of recombination, but is essential for the intermediate stage following the formation of DSBs but before the appearance of stable recombinants(10) . Most recently, human RAD52 protein has been shown to confer resistance to ionizing radiation and induce homologous recombination in monkey cells(29) .
Although interaction between yeast RAD52 and RAD51 proteins has been observed both in vitro(10) and in vivo(11, 12) , two observations prompted us to study the interaction between human RAD51 and RAD52 proteins. First, while the human RAD51 protein is highly homologous to the yeast RAD51 protein (83%) throughout the whole polypeptide, the human and yeast RAD52 proteins show significant homology only in the amino-terminal one-third. The carboxyl-terminal two-thirds of human RAD52 has little or no homology with the yeast RAD52 protein(20, 30, 31) . However, it is the carboxyl terminus of yeast RAD52 that is involved in the interaction with RAD51(11, 12) . This raises the question of whether human RAD52 and RAD51 interact at all. Second, neither human RAD51 cDNA (20) nor yeast Kluyveromyces lactis RAD51 complements the Rad51 defect of yeast S. cerevisiae(12) . This suggests that the interaction between RAD51 and RAD52 via the carboxyl terminus of RAD52 may be species-specific.
In this study, we show that human RAD52 physically associates with human RAD51 in vitro and in vivo. Additionally, the region of the RAD52 protein that provides this interaction with RAD51 has been determined. This RAD52 region shows no homology with yeast S. cerevisiae RAD52. These results suggest that a species-specific interaction between human RAD51 and RAD52 may be important for homologous recombination and/or DSB repair in mammalian cells.
RAD51 protein was incubated in batch with the AminoLink resin coupled with either RAD52 or BSA. RAD51 was dialyzed against buffer E and concentrated by centrifugation through an Amicon Centricon 30 filter. The concentrated RAD51 (250 µg in 250 µl) was mixed with 0.25 ml (drained volume) of the AminoLink resin covalently linked to either RAD52 or BSA. After incubating the mixture for 2 h at 4 °C with gentle mixing, the slurry was applied to a polyethylene column. The column was washed with buffer E. The RAD51 were eluted from the column with a step gradient of increasing NaCl concentration; each step (1 ml) contained buffer E plus either 50 mM, 0.15 M, 0.25 M, or 0.5 M NaCl. All steps were carried out at 4 °C and a flow rate of 0.2 ml/min. Fractions of 200 µl were collected, the absorbance at 280 nm was measured, and aliquots were used for immunoblot analysis using anti-RAD51 or anti-BSA antibodies to confirm the presence of relevant proteins in each fraction.
The self-association of yeast RAD51 protein and the
region responsible for this activity have been reported(12) .
However, self-association of RAD52 protein has not been reported. A
comprehensive analysis of the human RAD52 self-association will be
reported elsewhere. ()Interestingly, we consistently
observed weaker interactions between pGAD52 and pGBT51 as compared to
interaction between pGBT52 and pGAD51. Plasmid pVA3 that contains mouse
p53 protein (amino acids 72-390)/Gal4-DB fusion, and pTD1 that
contains SV40 large T-antigen (amino acids 64-708)/Gal4-DA fusion
were used as experiment controls. Although p53 interacts with large
T-antigen, p53 (amino acids 72-390) and T-antigen (amino acids
64-708) did not interact with human RAD51 or RAD52 in this yeast
two-hybrid assay (Table 1).
Although the two hybrid data
indicated in vivo interaction of human RAD52 and RAD51
proteins in yeast cells, this interaction was relatively weak as
compared to the self-association of either protein (Table 1).
Therefore, another in vivo experiment was conducted to confirm
the association of RAD51 and RAD52. RAD51 and RAD52 were expressed
independently or simultaneously in insect cells (Fig. 1A) using recombinant baculoviruses. The RAD51
and RAD52 complex was immunoprecipitated with antibodies specific to
either RAD51 or RAD52 (Fig. 1B). The immunoprecipitates
prepared by anti-RAD51 antibody contained a polypeptide that
cross-reacted with the anti-RAD52 antibody. The immunoprecipitates
prepared with anti-RAD52 contained a polypeptide that cross-reacted
with the anti-RAD51 antibody. Furthermore, immunoprecipitation
experiments with both antibodies on S-labeled HeLa cell
extracts also indicated physical association of RAD51 and RAD52 (data
not shown). These results show that RAD51 and RAD52 interact with each
other in vivo.
Figure 1: Co-expression and co-immunoprecipitation of human RAD51 and RAD52 protein in Sf9 cells. A, a Coomassie Blue-stained gel. lane WT, Sf9 cells infected with wild type virus; lane 51, Sf9 cells infected with recombinant RAD51 virus; lane 52, Sf9 cells infected with recombinant RAD52 virus; lane 51/52, Sf9 cells coinfected with RAD51 and RAD52 recombinant viruses. B, immunoprecipitation of the human RAD51 and RAD52 complex in Sf9 cells co-transfected with RAD51 and RAD52 recombinant baculoviruses. Lane 1, immunoprecipitated with anti-RAD51 and immunoblotted with anti-RAD52 antibody; lane 2, immunoprecipitated with anti-RAD52 antibody and immunoblotted with anti-RAD51 antibody.
Figure 2: Overexpression and purification of RAD51 and RAD52. A, Coomassie Blue-stained total cell extracts of E. coli overexpressing RAD51 (lane 1) and RAD52 (lane 2). B, Coomassie Blue-stained purified RAD51 (2 µg, lane 1) and RAD52 (2.5 µg, lane 2) proteins. C, Western analysis of purified RAD51 (500 ng, lane 1) and RAD52 (500 ng, lane 2).
Figure 3: Human RAD52 protein affinity chromatography of human RAD51 protein. RAD51 protein was applied to AminoLink resin that had been coupled with either RAD52 or BSA as described under ``Materials and Methods.'' RAD51 protein was eluted from the column using 1-ml step gradients containing 0 mM, 50 mM, 150 mM, 250 mM, and 500 mM NaCl (arrows). Two hundred-µl fractions were collected, and the absorbances at 280 nm were determined.
As a control, an identical experiment was carried out with RAD51 protein using AminoLink gel, that had been coupled with BSA instead of RAD52 protein (Fig. 3). In this case, most of the RAD51 protein eluted in the absence of NaCl, with only a low level (less than 5%) bound to the column. Thus, the affinity of RAD51 for the resin coupled to RAD52 protein is due to an interaction of RAD51 with RAD52 protein and not to any nonspecific interactions between the RAD51 and the resin. Therefore, this experiment shows that the interaction between human RAD52 and RAD51 is direct.
Figure 4: Determination of human RAD52 protein region interacting with RAD51. A, SDS-polyacrylamide gel electrophoresis analysis of purified RAD52 deletion mutant proteins. Lane A, RAD52(1-418); B, RAD52(78-418); C, RAD52(168-418); D, RAD52(291-418); E, RAD52(330-418); F, RAD52(260-340); G, RAD52(270-330). B, schematic illustration of RAD52 deletion mutants and results of their interaction with AminoLink-RAD51. RAD52 deletion mutants were tested for interaction with RAD51 coupled to AminoLink resin as described under ``Materials and Methods.'' Plus or minus sign indicates interaction or no interaction with AminoLink-RAD51, respectively.
For the in vivo experiment, a series of truncated RAD52 cDNAs were fused to the Gal4-DB in vector pGBT9. These constructs were co-transfected with pGAD51 into SFY526 cells. lacZ activity resulting from the interaction of Gal4-DB/RAD52 fusion constructs with full size RAD51/Gal4-DA fusion protein was assayed. Table 2indicates that the amino acids 287-333 are responsible for the interaction in yeast cells. Based on the in vivo and in vitro results taken together, we conclude that the RAD51 interaction domain of human RAD52 protein resides in the region of 291-330.
The data presented in this study clearly indicate that human
RAD51 and RAD52 proteins physically associate both in vivo and in vitro. Similar results have been described for yeast RAD51
and RAD52 proteins(10, 11, 12) . The present
study further mapped the human RAD52 region that interacts with the
human RAD51 protein. A region of approximately 40 amino acids near the
carboxyl terminus of RAD52 is required for interaction with RAD51 in vitro. Previous reports have shown that the human RAD51
protein cannot complement the Rad51 defect in yeast S.
cerevisiae(10) , and yeast K. lactis RAD51 has
little ability to restore the Rad51 defect in yeast S.
cerevisiae(12) . Since the RAD51 interaction
region(291-330) of human RAD52 does not share homology with the
yeast RAD52 protein, the interaction between RAD51 and RAD52 appears to
be species-specific. This specificity does not appear to be determined
by the RAD51 protein, because RAD51 and its homologs are well
conserved. The RAD52 self-association region has been mapped to its
amino-terminal region, which is independent of the RAD51
interaction region. Therefore, we have demonstrated that the human
RAD52 protein has at least two domains responsible for protein-protein
interactions.
Self-association of human RAD51 is consistent with the
results seen in yeast(12) . This self-association may be
required for self-assembly of RAD51 on DNA to form nucleofilaments with
a characteristic regularity that has been observed
previously(27) . The homotypic interaction domain of human
RAD51 appears to be present near its amino terminus ()as
seen with yeast RAD51 protein(12) .
The yeast RAD51 protein
has been shown to exhibit the catalytic activities of strand exchange
and homologous pairing(26) . More recently, the human RAD51 has
been shown to bind to DNA and also form RecA-like nucleofilaments on
DNA(27) , suggesting the possibility that the human RAD51 also
possesses either or both homologous pairing and strand exchange
activities(29) . The yeast RAD52 protein can bind to both
single- and double-stranded DNAs in the absence of ATP and carries out
annealing of homologous single-stranded DNA(40) . It can also
promote the strand transfer reaction(40) . However, the
reaction was ATP-independent and had an efficiency only 5% of that of
the RecA protein(40) . Besides the heterotypic and homotypic
interactions of RAD52, there is no direct information about the
biochemical activities of the mammalian RAD52 protein to date. Although
it can be speculated that the RAD52 protein might modulate the
catalytic activities of RAD51 based upon the information presented in
this study showing the direct association of RAD52 and RAD51 in
vivo and in vitro. The catalytic activity of yeast RAD51
can be exhibited in the absence of RAD52 protein in
vitro. However, a finer tuning of these processes in vivo may require other proteins such as RAD52.
Overexpression of the RAD52 protein in monkey cells provides a positive
effect on homologous recombination and protection from ionizing
radiation(29) . These results may further support the notion of
the RAD52 protein as a positive modulator of RAD51 activities for DSB
repair in mammalian cells. However, more direct evidence for this
hypothesis needs to be drawn from biochemical experiments in the
presence of both proteins in vitro.