Interaction of human rad51 recombination protein with single-stranded DNA binding protein, RPA.

Replication protein A (RPA), a heterotrimeric single-stranded DNA binding protein, is required for recombination, and stimulates homologous pairing and DNA strand exchange promoted in vitro by human recombination protein HsRad51. Co-immunoprecipitation revealed that purified RPA interacts physically with HsRad51, as well as with HsDmc1, the homolog that is expressed specifically in meiosis. The interaction with HsRad51 was mediated by the 70 kDa subunit of RPA, and according to experiments with deletion mutants, this interaction required amino acid residues 169-326. In exponentially growing mammalian cells, 22% of nuclei showed foci of RPA protein and 1-2% showed foci of Rad51. After gamma-irradiation, the percentage of cells with RPA foci increased to approximately 50%, and those with Rad51 foci to 30%. All of the cells with foci of Rad51 had foci of RPA, and in those cells the two proteins co-localized in a high fraction of foci. The interactions of human RPA with Rad51, replication proteins and DNA are suited to the linking of recombination to replication.     

DNase I cleavage of branched DNA molecules

We report here a potentially useful signature of branched DNA structures. The base 5' to the branch and the five bases flanking the 3' side of the branch site are protected from cleavage by DNase I in both three- and four-arm branched DNA molecules. Our procedure is to measure the cleavage profile for each 5' -labeled strand in a control duplex and compare this with that of the same strand in a branched structure under conditions yielding less than one cut per strand. The resulting cleavage pattern in an immobile four-arm junction is roughly 2-fold symmetric, consistent with the pattern of Fe(II).EDTA-induced cleavage that has been observed previously. In the three-arm junction, the DNase I cleavage pattern is asymmetric, indicating lack of 3-fold symmetry. A variable pattern of protection occurs to the 5' side of the branch in some strands only for both three- and four-arm junctions, extending 2-4 residues 5' to the branch.     

Interactions of human ribosomal protein S3 with intact and damaged DNA

Human S3 (hS3) is a structural component of the ribosome and, in addition to its role in translation, possesses apurinic/apyrimidinic (AP) lyase activity, characteristic of DNA repair enzymes. Recombinant hS3 was isolated from inclusion bodies, refolded under different conditions, and tested for the ability to bind and cleave oligodeoxyribonucleotide substrates with various lesions abundant in genomic DNA: AP sites, uracil, 8-oxoguanine, 8-oxoadenine, 5,6-dihydrouracil, and hypoxanthine. It was found that hS3 is capable of cleaving AP sites via the β-elimination mechanism, producing a Schiff base covalent intermediate, but cannot cleave substrates with the other lesions. Refolding in the presence of Fe²⁺ and S^2? did not increase hS3 activity, suggesting the absence of an iron-sulfur cluster. The binding of hS3 with DNA ligands containing oxidized or deaminated bases was less efficient than with intact DNA. It was assumed that the catalytic activity of hS3 towards AP sites is most likely unimportant for global DNA repair in vivo, but is possibly involved in repairing DNA sites in certain genome regions.     

Interactions of human ribosomal protein S3 with undamaged and damaged DNA

Human S3 protein (hS3) is a structural component of the ribosome, which, in addition to its role in translation, possesses activities typical of some DNA repair enzymes. Recombinant hS3 purified from inclusion bodies and refolded under different conditions was investigated for its ability to bind and cleave oligodeoxyribonucleotide substrates containing different lesions abundant in cellular DNA (apurine/apyrimidine sites, uracil, 8-oxoguanine, 8-oxoadenine, 5,6-dihydrouracil, hypoxanthine). hS3 catalyzed cleavage of apurine/apyrimidine sites through beta-elimination mechanism forming a transient Schiff base covalent intermediate, but did not cleave substrates containing other lesions. Refolding of hS3 in the presence of Fe2+ and S2- ions did not increase its activity, despite the earlier suggestions that this protein could contain an iron-sulfur cluster. Binding of hS3 to DNA ligands containing oxidized and deaminated bases was less efficient than its binding to undamaged DNA. Therefore, the activity of hS3 on apurine/apyrimidine sites is not likely to be involved in the global in vivo DNA repair but could have a role in the repair in some specific locations in the genome.     

Interactions between branched DNAs and peptide inhibitors of DNA repair

The RecG helicase of Escherichia coli unwinds both Holliday junction (HJ) and replication fork DNA substrates. Our lab previously identified and characterized peptides (WRWYCR and KWWCRW) that block the activity of RecG on these substrates. We determined that the peptides bind HJ DNA and prevent the binding of RecG. Herein, we present further evidence that the peptides are competitive inhibitors of RecG binding to its substrates. We have generated structural models of interactions between WRWYCR and a junction substrate. Using the fluorescent probe 2-aminopurine, we show that inhibitors interact with highest affinity with HJs (K_d = 14 nM) and ~4- to 9-fold more weakly with replication fork substrates. The fluorescence assay results agree with the structural model, and predict the molecular basis for interactions between HJ-trapping peptides and branched DNA molecules. Specifically, aromatic amino acids in the peptides stack with bases at the center of the DNA substrates. These interactions are stabilized by hydrogen bonds to the DNA and by intrapeptide interactions. These peptides inhibit several proteins involved in DNA repair in addition to RecG, have been useful as tools to dissect recombination, and possess antibiotic activity. Greater understanding of the peptides’ mechanism of action will further increase their utility.     

Visualization of Rad54, a chromatin remodeling protein, translocating on single DNA molecules

Rad54 protein plays an important role in the recombinational repair of double-strand DNA (dsDNA) breaks. It is a dsDNA-dependent ATPase that belongs to the Swi2/Snf2 family of chromatin-remodeling proteins. Rad54 remodels (1) DNA structure, (2) chromatin structure, and (3) Rad51-dsDNA complexes. These abilities imply that Rad54 moves along DNA. Here, we provide direct evidence of Rad54 translocation by visualizing its movement along single molecules of dsDNA. When compared to the remodeling processes, translocation is unexpectedly rapid, occurring at 301 +/- 22 bp/s at 25 degrees C. Rad54 binds randomly along the dsDNA and moves in either of the two possible directions with a velocity dependent on ATP concentration (K(m) = 97 +/- 28 microM). Movement is also surprisingly processive: the average distance traveled is approximately 11,500 bp, with molecules traversing up to 32,000 bp before stopping. The mechanistic implications of this vigorous Rad54 translocase activity in chromatin and protein-DNA complex remodeling are discussed.     

Genome instability in rad54 mutants of Saccharomyces cerevisiae

The RAD54 gene of Saccharomyces cerevisiae encodes a conserved dsDNA-dependent ATPase of the Swi2/Snf2 family with a specialized function during recombinational DNA repair. Here we analyzed the consequences of the loss of Rad54 function in vegetative (mitotic) cells. Mutants in RAD54 exhibited drastically reduced rates of spontaneous intragenic recombination but were proficient for spontaneous intergenic recombinant formation. The intergenic recombinants likely arose by a RAD54-independent pathway of break-induced replication. Significantly increased rates of spontaneous chromosome loss for diploid rad54/rad54 cells were identified in several independent assays. Inter estingly, the increase in chromosome loss appeared to depend on the presence of a homolog. In addition, the rate of complex genetic events involving chromosome loss were drastically increased in diploid rad54/rad54 cells. Together, these data suggest a role for Rad54 protein in the repair of spontaneous damage, where in the absence of Rad54 protein, homologous recombination is initiated but not properly terminated, leading to misrepair and chromosome loss.     

Correlation of biochemical properties with the oligomeric state of human rad52 protein

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.     

Analysis of fluorescence energy transfer in duplex and branched DNA molecules

Nonradiative fluorescence energy transfer (FET) is thought to be a highly sensitive measure of distance, occurring through a dipole coupling (Forster) mechanism in which the efficiency of FET depends on the inverse sixth power of the distance between fluorophores. The current work assesses the utility of FET for measuring distances in duplex and branched DNA molecules. The apparent efficiencies of FET between donor (fluorescein) and acceptor (eosin) fluorophores attached to opposite ends of oligonucleotide duplexes of varying length were determined; the results suggest that FET is a useful qualitative indicator of distance in DNA molecules. However, the apparent FET efficiency values cannot be fit to the Forster equation without the specification of highly extended DNA-to-fluorophore tethers and motionally restricted fluorophores, conditions that are unlikely to coexist. Three other lines of evidence further suggest that factors in addition to Forster transfer contribute to apparent FET in DNA: (1) The efficiency of FET appears to depend on the base sequence in some instances. (2) Donor fluorescence changes with the extent of thermally induced DNA melting in a sequence-dependent fashion, indicating dye-DNA interactions. (3) The distances between the ends of various pairwise combinations of arms of a DNA four-way junction do not vary as much as expected from previous work. Thus, the occurrence of any nondipolar effects on energy transfer in oligonucleotide systems must be defined before distances in DNA molecules can be quantified by using FET.     

Multiply branched DNA molecules from bacteriophage lambda: putative post-replicational repair DNA intermediates

Previous studies have shown that thymidine deprivation causes the formation of multiply branched molecules among bacteriophage lambda DNA replicative intermediates. In the present report, we present supporting evidence indicating that the induction of the SOS response is involved in this process. Moreover, close inspection of the DNA replicatives intermediates present in a recA deficient strain, shows an accumulation of gapped replicative intermediates. From these observations we postulate a model by which multiply branched DNA molecules may be intermediates or derived intermediates of a post-replicational repair pathway.     

A novel nucleic acid-binding protein that interacts with human rad51 recombinase.

Using the yeast two-hybrid system, we isolated a cDNA encoding a novel human protein, named Pir51, that strongly interacts with human Rad51 recombinase. Analysis in vitro confirmed the interaction between Rad51 and Pir51. Pir51 mRNA is expressed in a number of human organs, most notably in testis, thymus, colon and small intestine. The Pir51 gene locus was mapped to chromosome 12p13.1-13. 2 by fluorescence in situ hybridization. The Pir51 protein was expressed in Escherichia coli and purified to near homogeneity. Biochemical analysis shows that the Pir51 protein binds both single- and double-stranded DNA, and is capable of aggregating DNA. The protein also binds RNA. The Pir51 protein may represent a new member of the multiprotein complexes postulated to carry out homologous recombination and DNA repair in mammalian cells.     

Most chloroplast DNA of maize seedlings in linear molecules with defined ends and branched forms

We used pulsed-field gel electrophoresis, restriction fragment mapping, and fluorescence microscopy of individual DNA molecules to analyze the structure of chloroplast DNA (cpDNA) from shoots of ten to 14 day old maize seedlings. We find that most of the cpDNA is in linear and complex branched forms, with only 3-4% as circles. We find the ends of linear genomic monomers and head-to-tail (h-t) concatemers within inverted repeat sequences (IRs) near probable origins of replication, not at random sites as expected from broken circles. Our results predict two major and three minor populations of linear molecules, each with different ends and putative origins of replication. Our mapping data predict equimolar populations of h-t linear concatemeric molecules differing only in the relative orientation (inversion) of the single copy regions. We show how recombination during replication can produce h-t linear concatemers containing an inversion of single copy sequences that has for 20 years been attributed to recombinational flipping between IRs in a circular chromosome. We propose that replication is initiated predominantly on linear, not circular, DNA, producing multi-genomic branched chromosomes and that most replication involves strand invasion of internal regions by the ends of linear molecules, rather than the generally accepted D-loop-to-theta mechanism. We speculate that if the minor amount of cpDNA in circular form is useful to the plant, its contribution to chloroplast function does not depend on the circularity of these cpDNA molecules.     

Stimulation of Dmc1-mediated DNA strand exchange by the human Rad54B protein

The process of homologous recombination is indispensable for both meiotic and mitotic cell division, and is one of the major pathways for double-strand break (DSB) repair. The human Rad54B protein, which belongs to the SWI2/SNF2 protein family, plays a role in homologous recombination, and may function with the Dmc1 recombinase, a meiosis-specific Rad51 homolog. In the present study, we found that Rad54B enhanced the DNA strand-exchange activity of Dmc1 by stabilizing the Dmc1–single-stranded DNA (ssDNA) complex. Therefore, Rad54B may stimulate the Dmc1-mediated DNA strand exchange by stabilizing the nucleoprotein filament, which is formed on the ssDNA tails produced at DSB sites during homologous recombination.     

Lig4 and rad54 are required for repair of DNA double-strand breaks induced by P-element excision in Drosophila

Site-specific double-strand breaks (DSBs) were generated in the white gene located on the X chromosome of Drosophila by excision of the w(hd) P-element. To investigate the role of nonhomologous end joining (NHEJ) and homologous recombination (HR) in the repair of these breaks, the w(hd) P-element was mobilized in flies carrying mutant alleles of either lig4 or rad54. The survival of both lig4- and rad54-deficient males was reduced to 25% in comparison to the wild type, indicating that both NHEJ and HR are involved in the repair P-induced gaps in males. Survival of lig4-deficient females was not affected at all, implying that HR using the homologous chromosome as a template can partially compensate for the impaired NHEJ pathway. In rad54 mutant females survival was reduced to 70% after w(hd) excision. PCR analysis indicated that the undamaged homologous chromosome may compensate for the potential loss of the broken chromosome in rad54 mutant females after excision. Molecular analysis of the repair junctions revealed microhomology (2-8 bp)-dependent DSB repair in most products. In the absence of Lig4, the 8-bp target site duplication is used more frequently for repair. Our data indicate the presence of efficient alternative end-joining mechanisms, which partly depend on the presence of microhomology but do not require Lig4.     

Interactions of the human telomeric DNA with terbium-amino acid complexes

The human telomeric DNA can form four-stranded structures: the G-rich strand adopts a G-quadruplex conformation stabilized by G-quartets and the C-rich strand may fold into an I-motif based on intercalated C.C(+) base pairs. There is intense interests in the design and synthesis of compounds which can target telomeric DNA and inhibit the telomerase activity. Here we report the thermodynamic studies of the two newly synthesized terbium-amino acid complexes bound to the human telomeric G-quadruplex and I-motif DNA which were studied by means of UV-Visible, DNA meltings, fluorescence and circular dichroism. These two complexes can bind to the human telomeric DNA and have shown different features on DNA stability, binding stoichiometry, and sequence-dependent fluorescence enhancement. To our knowledge, this is the first report to show terbium-amino acid complexes can interact with the human telomeric DNA.     

Interactions of Escherichia coli replicative helicase PriA protein with single-stranded DNA

Quantitative analyses of the interactions of the Escherichia coli replicative helicase PriA protein with a single-stranded DNA have been performed, using the thermodynamically rigorous fluorescence titration technique. The analysis of the PriA helicase interactions with nonfluorescent, unmodified nucleic acids has been performed, using the macromolecular competition titration (MCT) method. Thermodynamic studies of the PriA helicase binding to ssDNA oligomers, as well as competition studies, show that independently of the type of nucleic acid base, as well as the salt concentration, the type of salt in solution, and nucleotide cofactors, the PriA helicase binds the ssDNA as a monomer. The enzyme binds the ssDNA with significant affinity in the absence of any nucleotide cofactors. Moreover, the presence of AMP-PNP diminishes the intrinsic affinity of the PriA protein for the ssDNA by a factor approximately 4, while ADP has no detectable effect. Analyses of the PriA interactions with different ssDNA oligomers, over a large range of nucleic acid concentrations, indicates that the enzyme has a single, strong ssDNA-binding site. The intrinsic affinities are salt-dependent. The formation of the helicase-ssDNA complexes is accompanied by a net release of 3-4 ions. The experiments have been performed with ssDNA oligomers encompassing the total site size of the helicase-ssDNA complex and with oligomers long enough to encompass only the ssDNA-binding site of the enzyme. The obtained results indicate that salt dependence of the intrinsic affinity results predominantly, if not exclusively, from the interactions of the ssDNA-binding site of the helicase with the nucleic acid. There is an anion effect on the studied interactions, which suggests that released ions originate from both the protein and the nucleic acid. Contrary to the intrinsic affinities, cooperative interactions between bound PriA molecules are accompanied by a net uptake of approximately 3 ions. The PriA protein shows preferential intrinsic affinity for pyrimidine ssDNA oligomers. In our standard conditions (pH 7.0, 10 degrees C, 100 mM NaCl), the intrinsic binding constant for the pyrimidine oligomers is approximately 1 order of magnitude higher than the intrinsic binding constant for the purine oligomers. The significance of these results for the mechanism of action of the PriA helicase is discussed.     

Structure of branched DNA molecules: gel retardation and atomic force microscopy studies.

DNA heteroduplexes as models for slipped strand DNA have been analyzed by polyacrylamide gel migration and atomic force microscopy (AFM). All heteroduplexes containing one hairpin or loop have reduced electrophoretic mobilities compared with that expected for their molecular weights. The retarded gel mobility correlates with the formation of a sharp kink detected by AFM. Increasing the hairpin length from 7 bp to 50 bp results in a monotonous decrease in gel mobility of heteroduplexes. This secondary retardation effect appears to depend only on the hairpin size since the AFM data show no dependence of the kink angle on the hairpin length. Heteroduplex isomers with a loop or hairpin in opposite strands migrate with distinct mobilities. Analysis of gel migration of heteroduplexes with altered hairpin orientations as well as of truncated heteroduplexes indicates that the difference in mobility is due to an inherent curvature in one of the long arms. This is confirmed by the end-to-end distance measurements from AFM images. In addition, significant variation of the end-to-end distances is consistent with a dynamic structure of heteroduplexes at the three-way junction. Double heteroduplexes containing one hairpin in each of the complementary strands also separate in a gel as two isomers. Their appearance in AFM showed a complicated pattern of flat representations of the three-dimensional structure and may indicate a certain degree of interaction between complementary parts of the hairpins that are several helical turns apart.