A transient assay for recombination demonstrates that Arabidopsis SNM1 and XRCC3 enhance non-homologous recombination

Replacement of endogenous genes by homologous recombination is rare in plants; the majority of genetic modifications are the result of transforming DNA molecules undergoing random genomic insertion by way of non-homologous recombination. Factors that affect chromatin remodeling and DNA repair are thought to have the potential to enhance the frequency of homologous recombination in plants. Conventional tools to study the frequencies of genetic recombination often rely on stable transformation-based approaches, with these systems being rarely capable of high-throughput or combinatorial analysis. We developed a series of vectors that use chemiluminescent (LUC and REN) reporter genes to assay the relative frequency of homologous and non-homologous recombination in plants. These transient assay vectors were used to screen 14 candidategenes for their effects on recombination frequencies in Nicotiana benthamiana plants. Over-expression of Arabidopsis genes with sequence similarity to SNM1 from yeast and XRCC3 from humans enhanced the frequency of non-homologous recombination when assayed using two different donor vectors. Transient N. benthamiana leaf systems were also used in an alternative assay for preliminary measurements of homologous recombination frequencies, which were found to be enhanced by over-expression of RAD52, MIM and RAD51 from yeast, as well as CHR24 from Arabidopsis. The findings for the assays described here are in line with previous studies that analyzed recombination frequencies using stable transformation. The assays we report have revealed functions in non-homologous recombination for the Arabidopsis SNM1 and XRCC3 genes, so the suppression of these genes' expression offers a potential means to enhance the gene targeting frequency in plants. Furthermore, our findings also indicate that plant gene targeting frequencies could be enhanced by over-expression of RAD52, MIM, CHR24, and RAD51 genes.

Phosphorylation of Exo1 modulates homologous recombination repair of DNA double-strand breaks

DNA double-strand break (DSB) repair via the homologous recombination pathway is a multi-stage process, which results in repair of the DSB without loss of genetic information or fidelity. One essential step in this process is the generation of extended single-stranded DNA (ssDNA) regions at the break site. This ssDNA serves to induce cell cycle checkpoints and is required for Rad51 mediated strand invasion of the sister chromatid. Here, we show that human Exonuclease 1 (Exo1) is required for the normal repair of DSBs by HR. Cells depleted of Exo1 show chromosomal instability and hypersensitivity to ionising radiation (IR) exposure. We find that Exo1 accumulates rapidly at DSBs and is required for the recruitment of RPA and Rad51 to sites of DSBs, suggesting a role for Exo1 in ssDNA generation. Interestingly, the phosphorylation of Exo1 by ATM appears to regulate the activity of Exo1 following resection, allowing optimal Rad51 loading and the completion of HR repair. These data establish a role for Exo1 in resection of DSBs in human cells, highlighting the critical requirement of Exo1 for DSB repair via HR and thus the maintenance of genomic stability.

A tumour-derived mutant allele of XRCC2 preferentially suppresses homologous recombination at DNA replication forks

Homologous recombination repair (HRR) is required for both the repair of DNA double strand breaks (DSBs) and the maintenance of the integrity of DNA replication forks. To determine the effect of a mutant allele of the RAD51 paralog XRCC2 (342delT) found in an HRR-defective tumour cell line, 342delT was introduced into HRR proficient cells containing a recombination reporter substrate. In one set of transfectants, expression of 342delT conferred sensitivity to thymidine and mitomycin C and suppressed HRR induced at the recombination reporter by thymidine but not by DSBs. In a second set of transfectants, the expression of 342delT was accompanied by a decreased level of the full-length XRCC2. These cells were defective in the induction of HRR by either thymidine or DSBs. Thus 342delT suppresses recombination induced by thymidine in a dominant negative manner while recombination induced by DSBs appears to depend upon the level of XRCC2 as well as the expression of the mutant XRCC2 allele. These results suggest that HRR pathways responding to stalled replication forks or DSBs are genetically distinguishable. They further suggest a critical role for XRCC2 in HRR at replication forks, possibly in the loading of RAD51 onto gapped DNA.

Promotion of Homologous Recombination and Genomic Stability by RAD51AP1 via RAD51 Recombinase Enhancement

Homologous recombination (HR) repairs chromosome damage and is indispensable for tumor suppression in humans. RAD51 mediates the DNA strand-pairing step in HR. RAD51 associated protein 1 (RAD51AP1) is a RAD51-interacting protein whose function has remained elusive. Knockdown of RAD51AP1 in human cells by RNA interference engenders sensitivity to different types of genotoxic stress, and RAD51AP1 is epistatic to the HR protein XRCC3. Moreover, RAD51AP1-depleted cells are impaired for the recombinational repair of a DNA double-strand break and exhibit chromatid breaks both spontaneously and upon DNA-damaging treatment. Purified RAD51AP1 binds both dsDNA and a D loop structure and, only when able to interact with RAD51, greatly stimulates the RAD51-mediated D loop reaction. Biochemical and cytological results show that RAD51AP1 functions at a step subsequent to the assembly of the RAD51-ssDNA nucleoprotein filament. Our findings provide evidence that RAD51AP1 helps maintain genomic integrity via RAD51 recombinase enhancement.

Mechanistic insights into RAD51-associated protein 1 (RAD51AP1) action in homologous DNA repair

Homologous recombination catalyzed by the RAD51 recombinase is essential for maintaining genome integrity upon the induction of DNA double strand breaks and other DNA lesions. By enhancing the recombinase activity of RAD51, RAD51AP1 (RAD51-associated protein 1) serves a key role in homologous recombination-mediated chromosome damage repair. We show here that RAD51AP1 harbors two distinct DNA binding domains that are both needed for maximal protein activity under physiological conditions. We have finely mapped the two DNA binding domains in RAD51AP1 and generated mutant variants that are impaired in either or both of the DNA binding domains. Examination of these mutants reveals that both domains are indispensable for RAD51AP1 function in cells. These and other results illuminate the mechanistic basis of RAD51AP1 action in homologous DNA repair.

Characterization of two homologous disulfide bond systems involved in virulence factor Biogenesis in Uropathogenic Escherichia coli CFT073

Disulfide bond (DSB) formation is catalyzed by disulfide bond proteins and is critical for the proper folding and functioning of secreted and membrane-associated bacterial proteins. Uropathogenic Escherichia coli (UPEC) strains possess two paralogous disulfide bond systems: the well-characterized DsbAB system and the recently described DsbLI system. In the DsbAB system, the highly oxidizing DsbA protein introduces disulfide bonds into unfolded polypeptides by donating its redox-active disulfide and is in turn reoxidized by DsbB. DsbA has broad substrate specificity and reacts readily with reduced unfolded proteins entering the periplasm. The DsbLI system also comprises a functional redox pair; however, DsbL catalyzes the specific oxidative folding of the large periplasmic enzyme arylsulfate sulfotransferase (ASST). In this study, we characterized the DsbLI system of the prototypic UPEC strain CFT073 and examined the contributions of the DsbAB and DsbLI systems to the production of functional flagella as well as type 1 and P fimbriae. The DsbLI system was able to catalyze disulfide bond formation in several well-defined DsbA targets when provided in trans on a multicopy plasmid. In a mouse urinary tract infection model, the isogenic dsbAB deletion mutant of CFT073 was severely attenuated, while deletion of dsbLI or assT did not affect colonization.

Analysis on conservation of disulphide bonds and their structural features in homologous protein domain families

Background: Disulphide bridges are well known to play key roles in stability, folding and functions of proteins. Introduction or deletion of disulphides by site-directed mutagenesis have produced varying effects on stability and folding depending upon the protein and location of disulphide in the 3-D structure. Given the lack of complete understanding it is worthwhile to learn from an analysis of extent of conservation of disulphides in homologous proteins. We have also addressed the question of what structural interactions replaces a disulphide in a homologue in another homologue. Results: Using a dataset involving 34,752 pairwise comparisons of homologous protein domains corresponding to 300 protein domain families of known 3-D structures, we provide a comprehensive analysis of extent of conservation of disulphide bridges and their structural features. We report that only 54% of all the disulphide bonds compared between the homologous pairs are conserved, even if, a small fraction of the non-conserved disulphides do include cytoplasmic proteins. Also, only about one fourth of the distinct disulphides are conserved in all the members in protein families. We note that while conservation of disulphide is common in many families, disulphide bond mutations are quite prevalent. Interestingly, we note that there is no clear relationship between sequence identity between two homologous proteins and disulphide bond conservation. Our analysis on structural features at the sites where cysteines forming disulphide in one homologue are replaced by non-Cys residues show that the elimination of a disulphide in a homologue need not always result in stabilizing interactions between equivalent residues. Conclusion: We observe that in the homologous proteins, disulphide bonds are conserved only to a modest extent. Very interestingly, we note that extent of conservation of disulphide in homologous proteins is unrelated to the overall sequence identity between homologues. The non-conserved disulphides are often associated with variable structural features that were recruited to be associated with differentiation or specialisation of protein function.

Evolution of three-dimensional character across the Lan+1NinO3n+1 homologous series with increase in n1

Electrical and magnetic properties of La3Ni2O7 and La4Ni3O10 have been investigated in comparison with those of La2NiO4, LaNiO3, and LaSrNiO4. The results suggest an increasing 3-dimensional character across the homologous series Lan+1NinO3n+1 with increase in n. Accordingly, the electrical resistivity decreases in the order La3Ni2O7, La4Ni3O10, and LaNiO3 and this trend is suggested to be related to the percolation threshold. Magnetic properties of these oxides also show some interesting trends across the series.

Structural adaptability in the system Bi2O3 - WO3 : Higher members of the homologous series Bi2WnO3n+3

The ultramicrostructure of phases with n = 1, 2 and 3 in the hypothetical series Bi2WnO3n+3 has been investigated by high resolution electron microscopy and energy dispersive X-ray emission spectroscopy. For n = 1 and 2, well ordered phases with the predicted compositions have been obtained, but for n = 3, a severely disordered assemblage containing intergrowths of the two known structures and strips of the n = 3 member is produced. No evidence for ordered structures with n > 2 has yet been obtained.

Structural adaptability in the system Bi2O3 --- WO3 : Higher members of the homologous series Bi2WnO3n+3

The ultramicrostructure of phases with n = 1, 2 and 3 in the hypothetical series Bi2WnO3n+3 has been investigated by high resolution electron microscopy and energy dispersive X-ray emission spectroscopy. For n = 1 and 2, well ordered phases with the predicted compositions have been obtained, but for n = 3, a severely disordered assemblage containing intergrowths of the two known structures and strips of the n = 3 member is produced. No evidence for ordered structures with n > 2 has yet been obtained.

RecA Protein of Mycobacterium tuberculosis Possesses pH-Dependent Homologous DNA Pairing and Strand Exchange Activities: Implications for Allele Exchange in Mycobacteria

To gain insights into inefficient allele exchange in mycobacteria, we compared homologous pairing and strand exchange reactions promoted by RecA protein of Mycobacterium tuberculosis to those of Escherichia coli RecA protein. The extent of single-stranded binding protein (SSB)-stimulated formation of joint molecules by MtRecA was similar to that of EcRecA over a wide range of pH values. In contrast, strand exchange promoted by MtRecA was inhibited around neutral pH due to the formation of DNA networks. At higher pH, MtRecA was able to overcome this constraint and, consequently, displayed optimal strand exchange activity. Order of addition experiments suggested that SSB, when added after MtRecA, was vital for strand exchange. Significantly, with shorter duplex DNA, MtRecA promoted efficient strand exchange without network formation in a pH-independent fashion. Increase in the length of duplex DNA led to incomplete strand exchange with concomitant rise in the formation of intermediates and networks in a pH-dependent manner. Treatment of purified networks with S1 nuclease liberated linear duplex DNA and products, consistent with a model in which the networks are formed by the invasion of hybrid DNA by the displaced linear single-stranded DNA. Titration of strand exchange reactions with ATP or salt distinguished a condition under which the formation of networks was blocked, but strand exchange was not significantly affected. We discuss how these results relate to inefficient allele exchange in mycobacteria.

Use of structure-directed DNA ligands to probe the binding of recA protein to narrow and wide grooves of DNA and on its ability to promote homologous pairing

We have used circular dichroism and structure-directed drugs to identify the role of structural features, wide and narrow grooves in particular, required for the cooperative polymerization, recognition of homologous sequences, and the formation of joint molecules promoted by recA protein. The path of cooperative polymerization of recA protein was deduced by its ability to cause quantitative displacement of distamycin from the narrow groove of duplex DNA. By contrast, methyl green bound to the wide groove was retained by the nucleoprotein filaments comprised of recA protein-DNA. Further, the mode of binding of these ligands and recA protein to DNA was confirmed by DNaseI digestion. More importantly, the formation of joint molecules was prevented by distamycin in the narrow groove while methyl green in the wide groove had no adverse effect. Intriguingly, distamycin interfered with the production of coaggregates between nucleoprotein filaments of recA protein-M13 ssDNA and naked linear M13 duplex DNA, but not with linear phi X174 duplex DNA. Thus, these data, in conjunction with molecular modeling, suggest that the narrow grooves of duplex DNA provide the fundamental framework required for the cooperative polymerization of recA protein and alignment of homologous sequences. These findings and their significance are discussed in relation to models of homologous pairing between two intertwined DNA molecules.

Recognition and alignment of homologous DNA sequences between minichromosomes and single-stranded DNA promoted by RecA protein

The incorporation of DNA into nucleosomes and higher-order forms of chromatin in vivo creates difficulties with respect to its accessibility for cellular functions such as transcription, replication, repair and recombination. To understand the role of chromatin structure in the process of homologous recombination, we have studied the interaction of nucleoprotein filaments, comprised of RecA protein and ssDNA, with minichromosomes. Using this paradigm, we have addressed how chromatin structure affects the search for homologous DNA sequences, and attempted to distinguish between two mutually exclusive models of DNA-DNA pairing mechanisms. Paradoxically, we found that the search for homologous sequences, as monitored by unwinding of homologous or heterologous duplex DNA, was facilitated by nucleosomes, with no discernible effect on homologous pairing. More importantly, unwinding of minichromosomes required the interaction of nucleoprotein filaments and led to the accumulation of circular duplex DNA sensitive to nuclease P1. Competition experiments indicated that chromatin templates and naked DNA served as equally efficient targets for homologous pairing. These and other findings suggest that nucleosomes do not impede but rather facilitate the search for homologous sequences and establish, in accordance with one proposed model, that unwinding of duplex DNA precedes alignment of homologous sequences at the level of chromatin. The potential application of this model to investigate the role of chromosomal proteins in the alignment of homologous sequences in the context of cellular recombination is considered.

DNA-induced conformational changes in RecA protein. Evidence for structural heterogeneity among nucleoprotein filaments and implications for homologous pairing

We have used circular dichroism as a probe to characterize the solution conformational changes in RecA protein upon binding to DNA. This approach revealed that RecA protein acquires significant amounts of alpha-helix upon interaction with DNA. These observations, consistent with the data from crystal structure (Story, R. M., Weber, I., and Steitz, T. (1992) Nature 355, 318-325), support the notion that some basic domains including the DNA binding motifs of RecA protein are unstructured and might contribute to the formation of alpha-helix. A comparison of nucleoprotein filaments comprised of RecA protein and a variety of DNA substrates revealed important structural heterogeneity. The most significant difference was observed with poly(dG). poly(dC) and related polymers, rich in GC sequences, which induced minimal amounts of alpha-helix in RecA protein. The magnitude of induction of alpha-helix in RecA protein, which occurred concomitant with the production of ternary complexes, was 2-fold higher with homologous than heterologous duplex DNA. Most importantly, the stimulation of ATP hydrolysis by high salt coincided with that of the induction of alpha-helix in RecA protein. These conformational differences provide a basis for thinking about the biochemical and structural transitions that RecA protein experiences during the formal steps of presynapsis, recognition, and alignment of homologous sequences.