Multiplex PCR: use of heat-stable Thermus thermophilus RecA protein to minimize non-specific PCR products

In this paper we report that the inclusion of heat-resistant RecA protein from a thermophilic bacteria, Thermus thermophilus, and its cofactor (ATP) in PCR effectively eliminates non-specific PCR products. The effect of RecA protein, which catalyzes pairing between homologous DNA molecules with great fidelity in genetic recombination, is due to its promotion of precise priming in PCR (i.e. priming at sites where the primer sequence is completely complementary to that of the target sequence). In addition, the RecA protein substantially reduces the primer concentration required for PCR. These experimental results have led to the realization of multiplex PCR, which involves PCR for multiple sites in the same reaction mixture. We were able to successfully perform multiplex PCR with over a dozen reactions without affecting the amplification pattern of the PCR products

The double-stranded break-forming activity of plant SPO11s and a novel rice SPO11 revealed by a Drosophila bioassay

<p>Abstract</p> <p>Background</p> <p>SPO11 is a key protein for promoting meiotic recombination, by generating chromatin locus- and timing-specific DNA double-strand breaks (DSBs). The DSB activity of SPO11 was shown by genetic analyses, but whether SPO11 exerts DSB-forming activity by itself is still an unanswered question. DSB formation by SPO11 has not been detected by biochemical means, probably because of a lack of proper protein-folding, posttranslational modifications, and/or specific SPO11-interacting proteins required for this activity. In addition, plants have multiple SPO11-homologues.</p> <p>Results</p> <p>To determine whether SPO11 can cleave DNA by itself, and to identify which plant SPO11 homologue cleaves DNA, we developed a <it>Drosophila </it>bioassay system that detects the DSB signals generated by a plant SPO11 homologue expressed ectopically. We cytologically and genetically demonstrated the DSB activities of <it>Arabidopsis </it>AtSPO11-1 and AtSPO11-2, which are required for meiosis, in the absence of other plant proteins. Using this bioassay, we further found that a novel SPO11-homologue, OsSPO11D, which has no counterpart in <it>Arabidopsis</it>, displays prominent DSB-forming activity. Quantitative analyses of the rice SPO11 transcripts revealed the specific increase in OsSPO11D mRNA in the anthers containing meiotic pollen mother cells.</p> <p>Conclusions</p> <p>The <it>Drosophila </it>bioassay system successfully demonstrated that some plant SPO11 orthologues have intrinsic DSB activities. Furthermore, we identified a novel SPO11 homologue, OsSPO11D, with robust DSB activity and a possible meiotic function.</p

RecR forms a ring-like tetramer that encircles dsDNA by forming a complex with RecF

In the RecFOR pathway, the RecF and RecR proteins form a complex that binds to DNA and exerts multiple functions, including directing the loading of RecA onto single-stranded (ss) DNA regions near double-stranded (ds) DNA–ssDNA junctions and preventing it from forming a filament beyond the ssDNA region. However, neither the structure of the RecFR complex nor its DNA-binding mechanism was previously identified. Here, size-exclusion chromatography and small-angle X-ray scattering data indicate that Thermus thermophilus (tt) RecR binds to ttRecF to form a globular structure consisting of four ttRecR and two ttRecF monomers. In addition, a low resolution model shows a cavity in the central part of the complex, suggesting that ttRecR forms a ring-like tetramer inside the ttRecFR complex. Mutant ttRecR proteins lacking the N- or C-terminal interfaces that are required for tetramer formation are unable to form a complex with ttRecF. Furthermore, a ttRecFR complex containing the DNA-binding deficient ttRecR K23E/R27E double mutant, which contains mutations lying inside the ring, exhibits significantly reduced dsDNA binding. Thus, we propose that the ring-like ttRecR tetramer has a key role in tethering the ttRecFR complex onto dsDNA and that the ring structure may function as a clamp protein

The process of displacing the single-stranded DNA-binding protein from single-stranded DNA by RecO and RecR proteins

The regions of single-stranded (ss) DNA that result from DNA damage are immediately coated by the ssDNA-binding protein (SSB). RecF pathway proteins facilitate the displacement of SSB from ssDNA, allowing the RecA protein to form protein filaments on the ssDNA region, which facilitates the process of recombinational DNA repair. In this study, we examined the mechanism of SSB displacement from ssDNA using purified Thermus thermophilus RecF pathway proteins. To date, RecO and RecR are thought to act as the RecOR complex. However, our results indicate that RecO and RecR have distinct functions. We found that RecR binds both RecF and RecO, and that RecO binds RecR, SSB and ssDNA. The electron microscopic studies indicated that SSB is displaced from ssDNA by RecO. In addition, pull-down assays indicated that the displaced SSB still remains indirectly attached to ssDNA through its interaction with RecO in the RecO-ssDNA complex. In the presence of both SSB and RecO, the ssDNA-dependent ATPase activity of RecA was inhibited, but was restored by the addition of RecR. Interestingly, the interaction of RecR with RecO affected the ssDNA-binding properties of RecO. These results suggest a model of SSB displacement from the ssDNA by RecF pathway proteins

DIDS, a chemical compound that inhibits RAD51-mediated homologous pairing and strand exchange

RAD51, an essential eukaryotic DNA recombinase, promotes homologous pairing and strand exchange during homologous recombination and the recombinational repair of double strand breaks. Mutations that up- or down-regulate RAD51 gene expression have been identified in several tumors, suggesting that inappropriate expression of the RAD51 activity may cause tumorigenesis. To identify chemical compounds that affect the RAD51 activity, in the present study, we performed the RAD51-mediated strand exchange assay in the presence of 185 chemical compounds. We found that 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) efficiently inhibited the RAD51-mediated strand exchange. DIDS also inhibited the RAD51-mediated homologous pairing in the absence of RPA. A surface plasmon resonance analysis revealed that DIDS directly binds to RAD51. A gel mobility shift assay showed that DIDS significantly inhibited the DNA-binding activity of RAD51. Therefore, DIDS may bind near the DNA binding site(s) of RAD51 and compete with DNA for RAD51 binding

Transient and persistent phosphorylation of AMPA-type glutamate receptor subunits in cerebellar Purkinje cells

AbstractWe generated a polyclonal antibody, 12P3, specifically recognizing rat AMPA-type glutamate receptor (GluR) subunits phosphorylated at Ser-696 of GIuR2 or at the homologous sites in GIuRl, GIuR3, and GIuR4. Using 12P3, we demonstrate that a brief exposure of a rat cerebellar slice to AMPA leads to transient phosphorylation of the GluR subunits in Purkinje cell dendrites. Persistent phosphorylation over 30 min was obtained when exposure to AMPA was preceded by a 15 min perfusion of the slice with 8-bromo-cGMP, dibutyryl-cGMP, or calyculin A but not phorbol 12,13-diacetate. These results indicate that Ser-696 of GIuR2, or the corresponding sites in other AMPA receptor subunits, is a specific site at which phosphorylation takes place when AMPA-type GluRs are activated by agonists, especially under the influence of certain second messenger activities