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Homologous recombination (HR) plays a critical role in facilitating replication fork progression when the polymerase complex encounters a blocking DNA lesion, and it also serves as the primary mechanism for error-free repair of DNA double strand breaks. Rad51 is the central catalyst of HR in all eukaryotes, and to this point studies of human Rad51 have focused exclusively on events occurring within the nucleus. However, substantial amounts of HR proteins exist in the cytoplasm, yet the function of these protein pools has not been addressed. Here, we provide the first demonstration that Rad51 and the related HR proteins Rad51C and Xrcc3 exist in human mitochondria. We show stress-induced increases in both the mitochondrial levels of each protein and, importantly, the physical interaction between Rad51 and mitochondrial DNA (mtDNA). Depletion of Rad51, Rad51C, or Xrcc3 results in a dramatic decrease in mtDNA copy number as well as the complete suppression of a characteristic oxidative stress-induced copy number increase. Our results identify human mtDNA as a novel Rad51 substrate and reveal an important role for HR proteins in the maintenance of the human mitochondrial genome.

Exposure of cells to DNA-damaging agents results in a rapid increase in the formation of subnuclear complexes containing Rad51. To date, it has not been determined to what extent DNA damage-induced cytoplasmic to nuclear transport of Rad51 may contribute to this process. We have analyzed subcellular fractions of HeLa and HCT116 cells and found a significant increase in nuclear Rad51 levels following exposure to a modest dose of ionizing radiation (2 grays). We also observed a DNA damage-induced increase in nuclear Rad51 in the Brca2-defective cell line Capan-1. To address a possible Brca2-independent mechanism for Rad51 nuclear transport, we analyzed subcellular fractions for two other Rad51-interacting proteins, Rad51C and Xrcc3. Rad51C has a functional nuclear localization signal, and although we found that the subcellular distribution of Xrcc3 was not significantly affected by DNA damage, there was a damage-induced increase in nuclear Rad51C. Furthermore, RNA interference-mediated depletion of Rad51C in HeLa and Capan-1 cells resulted in lower steady-state levels of nuclear Rad51 as well as a diminished DNA damage-induced increase. Our results provide important insight into the cellular regulation of Rad51 nuclear entry and a role for Rad51C in this process.

The human Rad52 protein self-associates to form ring-shaped oligomers, as well as higher order complexes of these rings. We have shown previously that there are two experimentally separable self-association domains in HsRad52, one in the N terminus (residues 1-192) responsible for assembly of individual subunits into rings, and one in the C terminus (residues 218-418) responsible for higher order oligomerization of rings. Earlier studies suggest that the higher order complexes promote DNA end-joining, and others suggest that these complexes are relevant to in vivo Rad52 function. In this study we demonstrate that although inherent binding to single-stranded DNA depends on neither higher order complexes of Rad52 rings nor the ring-shaped oligomers themselves, higher order complexes are important for activities involving simultaneous interaction with more than one DNA molecule. This provides biochemical support for what may be an important in vivo function of Rad52.

The human RAD52 protein plays an important role in the earliest stages of chromosomal double-strand break repair via the homologous recombination pathway. Individual subunits of RAD52 self-associate into rings that can then form higher order complexes. RAD52 binds to double-strand DNA ends, and recent studies suggest that the higher order self-association of the rings promotes DNA end-joining. Earlier studies defined the self-association domain of RAD52 to a unique region in the N-terminal half of the protein. Here we show that there are in fact two experimentally separable self-association domains in RAD52. The N-terminal self-association domain mediates the assembly of monomers into rings, and the previously unidentified domain in the C-terminal half of the protein mediates higher order self-association of the rings.

Binding of ATP to the RecA protein induces a high affinity DNA binding required for activation of enzyme function. Screens for in vivo recombination and repressor cleavage activities show Gln194 to be intolerant of all substitutions. Analyses of three mutant proteins (Q194N, Q194E, and Q194A) show that although basal enzyme function is maintained, each protein no longer displays an ATP-induced increase in DNA binding affinity. High salt activation of RecA function is also disrupted by these mutations. In contrast, ATP-induced changes in the oligomeric structure of RecA are maintained in the mutant proteins. These results demonstrate that Gln194 is a critical "allosteric switch" for ATP-induced activation of RecA function but is not the exclusive mediator of ATP-induced changes in RecA.

The functional significance of residues in the RecA protein P-loop motif was assessed by analyzing 100 unique mutants with single amino acid substitutions in this region. Comparison of the effects on the LexA coprotease and recombination activities shows that Pro67 is unique among these residues because only at this position did we find substitutions that caused differential effects on these functions. One mutant, Pro67-->Trp, displays high constitutive coprotease activity and a moderate inhibitory effect on recombination functions. Glu and Asp substitutions result in low level constitutive coprotease activity but dramatically reduce recombination activity. The purified Pro67-->Trp protein shows a completely relaxed specificity for NTP cofactors in LexA cleavage assays and can use shorter length oligonucleotides as cofactors for cleavage of lambda cI repressor than can wild type RecA. Interestingly, both the mutant protein and wild type RecA can use very short oligonucleotides, e.g. (dA)6 and (dT)6, as cofactors for LexA cleavage. We have also found two mutations at position 67, which are completely defective for LexA coprotease activity in vivo but still maintain recombinational DNA repair (Pro67-->Lys) and homologous recombination (Pro67-->Lys and Pro67-->Arg) activities. These findings show that the recombination activities of RecA are mutationally separable from the coprotease function and that Pro67 is located in a functionally important position in the RecA structure.

Using a combinatorial cassette mutagenesis procedure we have introduced a large number of single and multiple amino acid substitutions into an area of the RecA protein defined by residues 152-159. This sequence overlaps the disordered loop 1 region (L1) in the RecA crystal structure which has been hypothesized to be involved in DNA binding. Assays for recombinational DNA repair and LexA coprotease activities identify Glu154 as the only one of these 8 residues which is critical to RecA function. Several other mutations observed at nearby residues support the identity of Glu154 as the most important of the 14 residues in the area defined by Pro151 to Met164. In addition, Gly157 and Glu158 appear to be hot spots for the occurrence of mutation-induced constitutive coprotease activity.

Assembly of RecA subunits into long, helical oligomers is required for its roles in recombinational DNA repair and homologous genetic recombination. The crystal structure of RecA reveals an extensive network of amino acid residues that lie at the subunit boundaries. We have introduced a large set of substitutions at 5 clustered residues, which are shown in the crystal structure to make specific contacts with positions in the neighboring monomer. We find that 3 of the 5 residues are important for RecA function (Lys216, Phe217, and Arg222), whereas the other 2 (Asn213 and Tyr218) are not. The patterns of functionally allowed substitutions provide insight into the chemical and steric constraints required at these positions.

We have covalently modified the recA protein from Escherichia coli with the photoaffinity ATP analog 8-azido-[alpha-32P]ATP (N3-ATP). Covalent attachment of N3-ATP to recA protein is dependent on native protein conformation and is shown to be specific for the site of ATP hydrolysis by the following criteria. (i) Binding of the probe to recA protein is inhibited by ATP and competitive inhibitors of its ATP hydrolytic activity, e.g. adenosine 5'-O-(thiotriphosphate), ADP, and UTP, but not by adenosine; (ii) N3-ATP is efficiently hydrolyzed by recA protein in the presence of single-stranded DNA; (iii) labeling of recA protein occurs at a single site as judged by two-dimensional thin-layer peptide mapping and high-performance liquid chromatography peptide separation. We have purified and identified a tryptic fragment, spanning amino acid residues 257-280, which contains the primary site of attachment of N3-ATP. This peptide is likely to be contained within the ATP hydrolytic site of recA protein.

Genetic, biochemical, and biophysical studies have begun to reveal details of the structures of Arc and Mnt and show that these repressors use residues at their N-terminal ends for operator recognition and binding. Some of the DNA contacts made by these residues have been identified, and this information together with NMR studies has permitted the construction of models of the DNA binding region. Although the accuracy of these models remains to be determined, it seems clear that Arc and Mnt are members of a new class of DNA-binding proteins.

We have covalently modified the recA protein from Escherichia coli with the adenine nucleotide analog 5'-p-fluorosulfonylbenzoyladenosine (5'-FSBA). The rate at which the protein is modified shows a sigmoidal dependence on the concentration of 5'-FSBA suggesting that binding of the analog is characterized by positive cooperativity. Covalent modification of the protein results in irreversible inactivation of its single-stranded DNA-dependent ATPase activity such that 100% inactivation is achieved when 25% of the enzyme monomers have been modified. Attachment of 5'-FSBA is specific for the ATP-binding site of recA protein as judged by the following criteria: (i) attachment of the affinity label to the protein appears to saturate at 1 mol of 5'-FSBA/mol of protein; (ii) binding of 5'-FSBA to recA protein is inhibited by ATP and competitive inhibitors of its ATP hydrolytic activity, e.g. adenosine-5'-O-(thiotriphosphate), ADP, UTP, and GTP, but not by adenosine; (iii) attachment of 5'-FSBA to the protein occurs at a single site as determined by high pressure liquid chromatography peptide separation. Following trypsin digestion of recA protein that had been covalently modified with [3H]5'-FSBA we isolated a single labeled peptide (T31) containing the exclusive site of 5'-FSBA attachment. A secondary proteolytic digestion was performed on both 5'-FSBA modified T31 and unmodified T31 using Staphylococcus aureus V8 protease, and by comparison of the amino acid compositions of the resulting peptides we identified Tyr-264 as the exclusive site of 5'-FSBA attachment in recA protein.

We have identified the amino acid substitutions in two mutant forms of the recA protein from Escherichia coli. The recA441 mutant, which shows constitutive expression of the recA-mediated SOS response at 42 degrees C, contains two amino acid substitutions, glutamic acid to lysine at residue 38 and isoleucine to valine at residue 298. The recA629 mutant is an unusual pseudorevertant of recA441 that is no longer capable of spontaneous expression of SOS functions at 42 degrees C. Purified recA629 protein is cold-labile for several of the wild-type enzymatic activities and is shown here to contain three amino acid substitutions, the two found in the recA441 protein at residues 38 and 298, as well as an aspartic acid-to-glycine change at residue 32. The mutation at residue 32 was verified by restriction digestion of the 5' region of the recA629 structural gene.

Single amino acid substitutions have been introduced throughout the N-terminal DNA binding region of the Mnt repressor, and the operator binding properties of the resulting mutant repressors have been assayed. These studies show that the side chains of Arg2, His6, Asn8, and Arg10 are critical for high affinity binding to operator DNA. Other side chains in the N-terminal region do not appear to play major roles in DNA recognition and binding. Specific alterations in the pattern of methylation protection afforded by the Arg2----Lys mutant protein suggest that Arg2 contacts the N7 groups of guanines 10 and 12 in the operator. In conjunction with previous results, these findings suggest that part of the N-terminal region of Mnt binds as an extended polypeptide strand within the major groove of the mnt operator.

The photoaffinity label 8-azidoadenosine 5'-triphosphate (N3-ATP) was used to covalently modify the recA protein from Escherichia coli within its ATP-binding site. We have previously demonstrated that N3-ATP modification of recA protein is specific for the ATP-binding site and have isolated a unique tryptic peptide (T31), spanning residues 257-280, that contains the exclusive site of attachment of this ATP analog (Knight, K. L., and McEntee, K. (1985) J. Biol. Chem. 260, 867-872). We performed a secondary proteolytic digestion of the [alpha-32P]N3-ATP-labeled T31 peptide using Staphylococcus aureus V8 protease and purified the resulting peptide fragments by high-pressure liquid chromatography (HPLC). Based on a comparison of the amino acid compositions of all purified fragments and sequence analysis of one labeled fragment we determined that Tyr-264 is the exclusive site of N3-ATP attachment in recA protein. Photoaffinity labeling of recA protein was also performed in the presence of single-stranded DNA. Following trypsin treatment and separation of peptides by HPLC we showed that tryptic peptide T31 contained the exclusive site of N3-ATP attachment. A secondary proteolytic digestion was performed on both [alpha-32P]N3ATP-modified T31 and unmodified T31 using alpha-chymotrypsin. Comparison of the HPLC profiles and amino acid compositions of the resulting fragments was consistent with Tyr-264 as the exclusive site of N3-ATP attachment to recA protein.