Jin Inoue

Improvements of rolling circle amplification (RCA) efficiency and accuracy using Thermus thermophilus SSB mutant protein

Rolling circle amplification (RCA) of plasmid or genomic DNA using random hexamers and bacteriophage phi29 DNA polymerase has become increasingly popular in the amplification of template DNA in DNA sequencing. We have found that the mutant protein of single-stranded DNA binding protein (SSB) from Thermus thermophilus (Tth) HB8 enhances the efficiency of amplification of DNA templates. In addition, the TthSSB mutant protein increased the specificity of phi29 DNA polymerase. We have overexpressed the native and mutant forms of TthSSB protein in Escherichia coli and purified them to homogeneity. In vitro, these proteins were found to bind specifically to single-stranded DNA. Addition of TthSSB mutant protein to RCA halved the elongation time required for phi29 DNA polymerase to synthesize DNA fragments in RCA. Furthermore, the presence of the TthSSB mutant protein essentially eliminates nonspecific DNA products in RCA reactions.

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.

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.

Identification of the RecR Toprim Domain as the Binding Site for both RecF and RecO A ROLE OF RecR IN RecFOR ASSEMBLY AT DOUBLE-STRANDED DNA-SINGLE-STRANDED DNA JUNCTIONS

The RecR protein forms complexes with RecF or RecO that direct the specific loading of RecA onto gapped DNA. However, the binding sites of RecF and RecO on RecR have yet to be identified. In this study, a Thermus thermophilus RecR dimer model was constructed by NMR analysis and homology modeling. NMR titration analysis suggested that the hairpin region of the helix-hairpin-helix motif in the cavity of the RecR dimer is a binding site for double-stranded DNA (dsDNA) and that the acidic cluster region of the Toprim domain is a RecO binding site. Mutations of Glu-84, Asp-88, and Glu-144 residues comprising that acidic cluster were generated. The E144A and E84A mutations decreased the binding affinity for RecO, but the D88A did not. Interestingly, the binding ability to RecF was abolished by E144A, suggesting that the region surrounding the RecR Glu-144 residue could be a binding site not only for RecO but also for RecF. Furthermore, RecR and RecF formed a 4:2 heterohexamer in solution that was unaffected by adding RecO, indicating a preference by RecR for RecF over RecO. The RecFR complex is considered to be involved in the recognition of the dsDNA-ssDNA junction, whereas RecO binds single-stranded DNA (ssDNA) and ssDNA-binding protein. Thus, the RecR Toprim domain may contribute to the RecO interaction with RecFR complexes at the dsDNA-ssDNA junction site during recombinational DNA repair mediated by the RecFOR.

A mechanism for single-stranded DNA-binding protein (SSB) displacement from single-stranded DNA upon SSB-RecO interaction.

Displacement of single-stranded DNA (ssDNA)-binding protein (SSB) from ssDNA is necessary for filament formation of RecA on ssDNA to initiate homologous recombination. The interaction between RecO and SSB is considered to be important for SSB displacement; however, the interaction has not been characterized at the atomic level. In this study, to clarify the mechanism underlying SSB displacement from ssDNA upon RecO binding, we examined the interaction between Thermus thermophilus RecO and cognate SSB by NMR analysis. We found that SSB interacts with the C-terminal positively charged region of RecO. Based on this result, we constructed some RecO mutants. The R127A mutant had considerably decreased binding affinity for SSB and could not anneal SSB-coated ssDNAs. Further, the mutant in the RecOR complex prevented the recovery of ssDNA-dependent ATPase activity of RecA from inhibition by SSB. These results indicated that the region surrounding Arg-127 is the binding site of SSB. We also performed NMR analysis using the C-terminal peptide of SSB and found that the acidic region of SSB is involved in the interaction with RecO, as seen in other protein-SSB interactions. Taken together with the findings of previous studies, we propose a model for SSB displacement from ssDNA where the acidic C-terminal region of SSB weakens the ssDNA binding affinity of SSB when the dynamics of the C-terminal region are suppressed by interactions with other proteins, including RecO.

Single-stranded DNA binding protein facilitates specific enrichment of circular DNA molecules using rolling circle amplification

Many techniques in molecular biology require the use of pure nucleic acids in general and circular DNA (plasmid or mitochondrial) in particular. We have developed a method to separate these circular molecules from a mixture containing different species of nucleic acids using rolling circle amplification (RCA). RCA of plasmid or genomic DNA using random hexamers and bacteriophage Phi29 DNA polymerase has become increasingly popular for the amplification of template DNA in DNA sequencing protocols. Recently, we reported that the mutant single-stranded DNA binding protein (SSB) from Thermus thermophilus (TthSSB) HB8 eliminates nonspecific DNA products in RCA reactions. We developed this method for separating circular nucleic acids from a mixture having different species of nucleic acids. Use of the mutant TthSSB resulted in an enhancement of plasmid or mitochondrial DNA content in the amplified product by approximately 500x. The use of mutant TthSSB not only promoted the amplification of circular target DNA over the background but also could be used to enhance the amplification of circular targets over linear targets.

A specific single-stranded DNA induces a distinct conformational change in the nucleoid-associated protein HU

In prokaryotic cells, genomic DNA forms an aggregated structure with various nucleoid-associated proteins (NAPs). The functions of genomic DNA are cooperatively modulated by NAPs, of which HU is considered to be one of the most important. HU binds double-stranded DNA (dsDNA) and serves as a structural modulator in the genome architecture. It plays important roles in diverse DNA functions, including replication, segregation, transcription and repair. Interestingly, it has been reported that HU also binds single-stranded DNA (ssDNA) regardless of sequence. However, structural analysis of HU with ssDNA has been lacking, and the functional relevance of this binding remains elusive. In this study, we found that ssDNA induced a significant change in the secondary structure of Thermus thermophilus HU (TtHU), as observed by analysis of circular dichroism spectra. Notably, this change in secondary structure was sequence specific, because the complementary ssDNA or dsDNA did not induce the change. Structural analysis using nuclear magnetic resonance confirmed that TtHU and this ssDNA formed a unique structure, which was different from the previously reported structure of HU in complex with dsDNA. Our data suggest that TtHU undergoes a distinct structural change when it associates with ssDNA of a specific sequence and subsequently exerts a yet-to-be-defined function.

Use of native gels to measure protein binding to SSB.

We describe a procedure to detect protein binding to SSB by polyacrylamide gel electrophoresis under non-denaturing conditions. As an example, we show the interaction of Thermus thermophilus (Tth) SSB with its cognate RecO protein. The interaction is detected as decay of the band corresponding to SSB by addition of RecO. We also demonstrate analysis of the RecO-RecR interaction as another example of this method.