Direct Quantitative Analysis of Multiple miRNAs (DQAMmiR)

BackgroundDeinococcus radiodurans R1 is one of the most radiation-resistant organisms known and is able to repair an unusually large amount of DNA damage without induced mutation. Single-stranded DNA-binding (SSB) protein is an essential protein in all organisms and is involved in DNA replication, recombination and repair. The published genomic sequence from Deinococcus radiodurans includes a putative single-stranded DNA-binding protein gene (ssb; DR0100) requiring a translational frameshift for synthesis of a complete SSB protein. The apparently tripartite gene has inspired considerable speculation in the literature about potentially novel frameshifting or RNA editing mechanisms. Immediately upstream of the ssb gene is another gene (DR0099) given an ssb-like annotation, but left unexplored.ResultsA segment of the Deinococcus radiodurans strain R1 genome encompassing the ssb gene has been re-sequenced, and two errors involving omitted guanine nucleotides have been documented. The corrected sequence incorporates both of the open reading frames designated DR0099 and DR0100 into one contiguous ssb open reading frame (ORF). The corrected gene requires no translational frameshifts and contains two predicted oligonucleotide/oligosaccharide-binding (OB) folds. The protein has been purified and its sequence is closely related to the Thermus thermophilus and Thermus aquaticus SSB proteins. Like the Thermus SSB proteins, the SSBDr functions as a homodimer. The Deinococcus radiodurans SSB homodimer stimulates Deinococcus radiodurans RecA protein and Escherichia coli RecA protein-promoted DNA three-strand exchange reactions with at least the same efficiency as the Escherichia coli SSB homotetramer.ConclusionsThe correct Deinococcus radiodurans ssb gene is a contiguous open reading frame that codes for the largest bacterial SSB monomer identified to date. The Deinococcus radiodurans SSB protein includes two OB folds per monomer and functions as a homodimer. The Deinococcus radiodurans SSB protein efficiently stimulates Deinococcus radiodurans RecA and also Escherichia coli RecA protein-promoted DNA strand exchange reactions. The identification and purification of Deinococcus radiodurans SSB protein not only allows for greater understanding of the SSB protein family but provides an essential yet previously missing player in the current efforts to understand the extraordinary DNA repair capacity of Deinococcus radiodurans.

Single Stranded DNA Translocases can Push Single Stranded DNA Binding Proteins along Single Stranded DNA

Dynamics of E. coli single stranded DNA binding (SSB) protein-DNA complexes

Single stranded DNA (ssDNA) binding proteins (SSBPs), are known key players of DNA damage response (DDR) pathway and play an essential role in stabilising fragile ssDNA generated during DNA replication, transcription and repair. The canonical SSBP is the heterotrimeric Replication Protein A (RPA) which is involved in a number of key cellular processes including replication and repair via Homologous Recombination (HR) in the course of DNA damage. Our lab recently described two new SSBPs, termed SSB1 and SSB2 (also known as NABP2/OBFC2B/SOSS-B1 and NABP1/OBFC2A/SOSSB-2, respectively) which form independent co-complexes with two additional proteins, the Integrator complex subunit 3 (INTS3) and the chromosome 9 open reading frame 80 (C9ORF80), a small acidic 104 residue polypeptide. Previously, we demonstrated that whilst Ssb1/Nabp2 KO in mouse caused perinatal lethality, Ssb2/Nabp1 KO did not lead to any phenotypic abnormalities. Interestingly, ablation of Ssb1 led to stabilisation of Ssb2 and vice-versa, indicating functional redundancy between these two proteins. This was recently demonstrated in-vivo by the generation of Ssb1 and Ssb2 (together referred as Ssb1/2) double-knockout (DKO) mice, which caused early embryonic lethality in a constitutive model and acute bone marrow failure and intestinal atrophy using the inducible Rosa26-CreERT2 system. To delineate the functional redundancy between these two proteins at the molecular level, we have generated inducible DKO mouse embryonic fibroblasts (MEFs) using the Rosa26-CreERT2 system, which will be described in the first research chapter. We found that cumulative loss of Ssb1/2 in the primary as well as SV40-immortalised MEFs led to acute proliferation arrest and cell death following TAM administration. This was associated with accumulation of genomic instability via endogenous replication stress. Although loss of Ssb1/2 in-vivo and in-vitro is associated with accumulation of R-loops, the overall DKO phenotype was not able to be rescued with overexpression of RNaseH1, which resolves R-loops. Additionally, we investigated the roles of Ssb1/2 following treatment with different DNA damaging agents to determine their roles in the DDR system. Interestingly, DDR signalling in DKO was normal following ionizing radiation, ultraviolet C and camptothecin but with hydroxyurea treatment that causes replication stress, we observed a delayed signalling response in DKO. Together, this chapter defines the phenotypic changes that take place in-vitro when Ssb1 and Ssb2 are deleted. The second research chapter describes our finding that loss of Ssb1 and Ssb2 together leads to reduced levels of several Integrator components and thus, has an equivalent profound effect on the misprocessing of the Sm- associated small nuclear RNAs (snRNAs) to that of the Integrator catalytic components- IntS9 and IntS11. Here, we show that upregulated snRNAs are not only misprocessed, but extend up to several hundred to a thousand base pairs past their native termination site, and are polyadenylated. Additionally, we demonstrate that loss of Ssb1/2 led to changes in the dynamics of alternative splicing, likely due to perturbation of the splicing machinery by aberrant snRNAs. We further show that a number of regulators of transcription and the cell cycle are affected by these changes, which might contribute to the loss of viability observed in DKO cells. Together, these findings reveal the critical role of Ssb1/2 and their association with the Integrator complex in regulating cellular proliferation and spliceosomal function. The third chapter of this thesis further investigates the intestinal atrophy observed upon loss of Ssb1 and Ssb2 in the DKO mice from our lab. For this, we have generated a small intestine (SI) specific Ssb1/2 DKO mouse- the VillinCreERT2 Ssb1flox/flox; Ssb2flox/flox model. This mouse model is a unique system to study the undefined roles of Ssb1/2 in the small intestine (SI) by bypassing the confounding effects of the bone marrow phenotype in the ubiquitous Rosa26-CreERT2 DKO model. We have found that loss of intestinal Ssb1/2 leads to exhaustion of the stem cells in the crypts, resulting in loss of the normal crypt-villus axis anatomy which causes acute morbidity within six days of induction. Interestingly, the stem cells are pushed to proliferate immediately after the loss of Ssb1 and Ssb2, followed by the exhaustion of these cells. This is demonstrated by sequential proliferation studies using the known thymidine analogue 5-bromo-2’deoxyuridine (BrdU) as well as quantitative reverse transcription polymerase chain reaction (qRT-PCR). Therefore through this model, we have demonstrated a fundamental role of Ssb1/2 in the maintenance of intestinal homeostasis. In conclusion, through the inducible abrogation of two SSBPs- Ssb1 and Ssb2 together, we have demonstrated several novel roles of these proteins in the maintenance of genomic stability both in-vitro and in-vivo that were previously masked in single KO studies. Further, we have defined the molecular mechanisms underlying the acute lethality observed upon abrogation of these two proteins.

Protein-DNA interactions play a critical role in many biological processes. However, the structural mechanisms underlying these interactions are not fully understood. DNA binding proteins can be classified into double-stranded DNA binding proteins (DSBs) and single-stranded DNA binding proteins (SSBs). Understanding the binding specificity of a DNA binding protein is helpful for the research of protein functions. Though there are some researches [1] on the SSB and DSB respectively, few attentions have been paid on investigating what makes SSB and DSB have such different ability of the specific binding. With the development of biotechnology, a large amount of proteins has been sequenced. However, SSBs have shown to have little sequence conservation [2]. Even DSBs involved in similar functions may have conserved subsequences, different kinds of DSBs with different functions seems to show few common subsequences. Therefore, it is hard to recognize SSB sequences from DSB sequences, or vice versa. In fact, up to Jan. 25, 2013, the Protein Data Bank (PDB) [3] contains 3391 structures for DNA binding proteins, among them only about 30% and 5% are annotated as DSBs and SSBs, respectively, and whether the remainders belong to DSBs or SSBs are still not very clear. Therefore, a computational method is required to annotate the DNA binding protein as DSB or SSB automatically. The surface of a protein is generally irregular, containing many clefts and grooves of varying shapes and sizes. Previous researches have shown that a large cleft can provide an increased opportunity for the protein to form interactions with other molecules, particularly small ligands [4]. Therefore, some researches used a particularly large and deep cleft to characterize the binding active sites of the proteins [5]. We guess that for DNA binding proteins, the cleft properties on the surface may also play important roles on the dsDNA/ssDNA binding specificity. In this work, we applied CAVER 3.0 package [6] to detect the clefts and the corresponding indexes of the largest clefts on the protein surfaces, to investigate whether they are possible to be used for distinguishing the potential interfaces between SSBs and DSBs. Concretely, we mainly got three indexes of the detected tunnels: length, curvature and bottleneck radius. Research results have shown that although the sequences of different SSBs are very different, there are well-conserved elements in the structures. That is, most SSBs contain one or more OB (oligonucleotide/oligosaccharide binding) - fold domains [2]. A typical OB-fold has a five-stranded beta-sheet coiled to form a closed beta-barrel. This barrel is capped by an alpha-helix located between the third and fourth strands. The OB-fold plays critical role in binding with ssDNA. Although it is hard to say that the OB-fold is unique for SSBs, we think that it should also be used as an important descriptor to distinguish SSBs from DSBs. Therefore, we use the protein structure alignment package TM-align [7] to compare its structure with each of the six OB-fold protein templates and use the maximal alignment score TM-score as the OB-fold feature of the protein. We aim to investigate the structural differences between collected SSBs and DSBs, and extract the structure-based features related to surface clefts and OB-folds, Based on which, we construct a computational model that can automatically classify the DNA protein as a DSB or SSB by using the widely used support vector machine (SVM), with prediction accuracy of HOLO-set 0.87, APO-set 0.83, and mixed-set 0.83, respectively. The promising performance suggests that our method will be useful in the protein function annotation and refinement. This work is supported by grant from the National Science Foundation of China (61272274); Program for New Century Excellent Talents in Universities (NCET-10-0644), the Open Research Fund of State Key Laboratory of Hybrid Ri ce (Wuhan University) (KF201301) and the Fundamental Research Funds for the Central Universities (No. 2012211020204).

The pharmacological actions of morphine and morphine-like drugs such as heroin are mediated primarily through the μ opioid receptor. Previously a single strand DNA element of the mouse μ opioid receptor gene (Oprm1) proximal promoter was found to be important for regulating Oprm1 in neuronal cells. To identify proteins binding to the single strand DNA element as potential regulators for Oprm1, affinity column chromatography with the single strand DNA element was performed using neuroblastoma NS20Y cells followed by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. We identified five poly(C)-binding proteins: heterogeneous nuclear ribonucleoprotein (hnRNP) K, α-complex proteins (αCP) αCP1, αCP2, αCP2-KL, and αCP3. Binding of these proteins to the single strand DNA element of Oprm1 was sequence-specific as confirmed by supershift assays. In cotransfection studies, hnRNP K, αCP1, αCP2, and αCP2-KL activated the Oprm1 promoter activity, whereas αCP3 acted as a repressor. Ectopic expression of hnRNP K, αCP1, αCP2, and αCP2-KL also led to activation of the endogenous Oprm1 transcripts, and αCP3 repressed endogenous Oprm1 transcripts. We demonstrate novel roles as transcriptional regulators in Oprm1 regulation for hnRNP K and αCP binding to the single strand DNA element.

Ligand induced stabilization of the melting temperature of the HSV-1 single-strand DNA binding protein using the thermal shift assay

The recA730 mutation results in constitutive SOS and prophage induction. We examined biochemical properties of recA730 protein in an effort to explain the constitutive activity observed in recA730 strains. We find that recA730 protein is more proficient than the wild-type recA protein in the competition with single-stranded DNA binding protein (SSB protein) for single-stranded DNA (ssDNA) binding sites. Because an increased aptitude in the competition with SSB protein has been previously reported for recA441 protein and recA803 protein, we directly compared their in vitro activities with those of recA730 protein. At low magnesium ion concentration, both ATP hydrolysis and lexA protein cleavage experiments demonstrate that these recA proteins displace SSB protein from ssDNA in a manner consistent with their in vivo repressor cleavage activity, i.e. recA730 protein > recA441 protein > recA803 protein > recAwt protein. Additionally, a correlation exists between the proficiency of the recA proteins in SSB protein displacement and their rate of association with ssDNA. We propose that an increased rate of association with ssDNA allows recA730 protein to displace SSB protein from the ssDNA that occurs naturally in Escherichia coli and thereby to become activated for the repressor cleavage that leads to SOS induction. RecA441 protein is similarly activated for repressor cleavage; however, in this case, significant SSB protein displacement occurs only at elevated temperature. At physiological magnesium ion concentration, we argue that recA803 protein and wild-type recA protein do not displace sufficient SSB protein from ssDNA to constitutively induce the SOS response.

Single-stranded DNA-binding protein in Bacteria and replication protein A (RPA) in Eukarya play crucial roles in DNA replication, repair, and recombination processes. We identified an RPA complex from the hyperthermophilic archaeon, Pyrococcus furiosus. Unlike the single-peptide RPAs from the methanogenic archaea, Methanococcus jannaschii and Methanothermobacter thermoautotrophicus, P. furiosus RPA (PfuRPA) exists as a stable hetero-oligomeric complex consisting of three subunits, RPA41, RPA14, and RPA32. The amino acid sequence of RPA41 has some similarity to those of the eukaryotic RPA70 subunit and the M. jannaschii RPA. On the other hand, RPA14 and RPA32 do not share homology with any known open reading frames from Bacteria and Eukarya. However, six of eight archaea, whose total genome sequences have been published, have the open reading frame homologous to RPA32. The PfuRPA complex, but not each subunit alone, specifically bound to a single-stranded DNA and clearly enhanced the efficiency of an in vitro strand-exchange reaction by the P. furiosus RadA protein. Moreover, immunoprecipitation analyses showed that PfuRPA interacts with the recombination proteins, RadA and Hjc, as well as replication proteins, DNA polymerases, primase, proliferating cell nuclear antigen, and replication factor C in P. furiosus cells. These results indicate that PfuRPA plays important roles in the homologous DNA recombination in P. furiosus.

Here, we describe a new fluorescence polarization aptamer assay (FPAA) strategy which is based on the use of the single-stranded DNA binding (SSB) protein from Escherichia coli as a strong FP signal enhancer tool. This approach relied on the unique ability of the SSB protein to bind the nucleic acid aptamer in its free state but not in its target-bound folded one. Such a feature was exploited by using the antiadenosine (Ade)-DNA aptamer (Apt-A) as a model functional nucleic acid. Two fluorophores (fluorescein and Texas Red) were introduced into different sites of Apt-A to design a dozen fluorescent tracers. In the absence of the Ade target, the binding of the labeled aptamers to SSB governed a very high fluorescence anisotropy increase (in the 0.130-0.200 range) as the consequence of (i) the large global diffusion difference between the free and SSB-bound tracers and (ii) the restricted movement of the dye in the SSB-bound state. When the analyte was introduced into the reaction system, the formation of the folded tertiary structure of the Ade-Apt-A complex triggered the release of the labeled nucleic acids from the protein, leading to a strong decrease in the fluorescence anisotropy. The key factors involved in the fluorescence anisotropy change were considered through the development of a competitive displacement model, and the optimal tracer candidate was selected for the Ade assay under buffer and realistic (diluted human serum) conditions. The SSB-assisted principle was found to operate also with another aptamer system, i.e., the antiargininamide DNA aptamer, and a different biosensing configuration, i.e., the sandwich-like design, suggesting the broad usefulness of the present approach. This sensing platform allowed generation of a fluorescence anisotropy signal for aptamer probes which did not operate under the direct format and greatly improved the assay response relative to that of the most previously reported small target FPAA.

We have constructed a mutant form of the RecBCD enzyme from Escherichia coli with a lysine to glutamine change in the consensus ATP-binding sequence in the RecD subunit (Korangy, F., and Julin, D.A. (1992a, 1992b) J. Biol. Chem., 1727-1732; 1733-1740). We compare here the kinetics of double-stranded DNA-dependent ATP hydrolysis by the mutant (RecBCD-K177Q) and wild-type enzymes. We included heparin to trap enzyme not bound to DNA, or the single-stranded DNA-binding (SSB) protein from Escherichia coli to prevent the enzyme from binding to single-stranded DNA products and partially single-stranded reaction intermediates. The ATP hydrolysis kinetics in either case show a rapid burst phase followed by a slower second phase. The wild-type enzyme hydrolyzes an amount of ATP about equal to the DNA nucleotide concentration in the rapid phase. The amount of ATP hydrolyzed by the RecBCD-K177Q enzyme in the burst is about 8-10-fold lower than the wild-type, in the presence of either heparin or SSB. The burst magnitude of the wild-type enzyme with heparin is proportional to the size of the DNA from about 1,420 to 22,400 base pairs whereas that of the mutant is independent of the DNA size. The wild-type enzyme completely degrades a 6,250-base pair DNA substrate with no partially degraded molecules visible on agarose gels. RecBCD-K177Q enzyme reaction mixtures in the presence of SSB protein contain a heterogeneous mixture of partially degraded molecules of 2,000-5,000 base pairs. These results indicate that the RecBCD-K177Q enzyme is less processive than the wild-type enzyme.

Binding of the Single-Stranded DNA Binding Protein (gp32) of T4 Bacteriophage Induces Position-Specific Local Conformational Changes in DNA Lattices that can be Monitored by Fluorescent Probes

Recombination directionality factors (RDFs) for large serine integrases (LSIs) are cofactor proteins that control the directionality of recombination to favour excision over insertion. Although RDFs are predicted to bind their cognate LSIs in similar ways, there is no overall common structural theme across LSI RDFs, leading to the suggestion that some of them may be moonlighting proteins with other primary functions. To test this hypothesis, we searched for characterized proteins with structures similar to the predicted structures of known RDFs. Our search shows that the RDFs for two LSIs, TG1 integrase and Bxb1 integrase, show high similarities to a single-stranded DNA binding (SSB) protein and an editing exonuclease, respectively. We present experimental data to show that Bxb1 RDF is probably an exonuclease and TG1 RDF is a functional SSB protein. We used mutational analysis to validate the integrase-RDF interface predicted by AlphaFold2 multimer for TG1 integrase and its RDF, and establish that control of recombination directionality is mediated via protein-protein interaction at the junction of recombinase's second DNA binding domain and the base of the coiled-coil domain.

The functional role of Zn(II) binding by T4 gene 32 protein (gp32), a single-stranded DNA-binding protein, has been investigated by assessing the capacity of a well-characterized metal-free gp32 derivative to function in vitro as an accessory protein of T4 uvsX-catalyzed homologous pairing. Metal-free gp32 was prepared upon reaction of cysteine thiolates with methylmethanethiol-sulfonate to form the mixed disulfide Cys-SSCH3 or S-methylated species. Far and near ultraviolet circular dichroism spectroscopy suggest a moderate but easily detected change in the far UV region, accompanied by only a minor alteration in the near UV region, relative to the Zn(II)-containing protein. Restoration of the wild-type spectral features is accomplished upon the addition of 2 mM dithiothreitol and excess Zn(II) but not dithiothreitol alone. Unlike wild-type gp32, apo S-methylated gp32 shows weak binding to the recombination substrate, single-stranded M13mp19, and fails to stimulate homologous pairing with a linear M13mp19 duplex substrate by uvsX protein. Complete reactivation of the apo S-methylated protein as a recombination-accessory protein is achievable in situ in the presence of reducing agent and sufficient exogenous Zn(II), but not one or the other alone. Analogous results are obtained with S-methylated C166S (Cys166-->Ser) gp32, revealing that only the metal-liganding cysteines participate in the reconstitution. These findings suggest that formation of the Zn(II) chelate is directly linked to single-stranded DNA binding and functional efficacy of gp32 in DNA metabolism.

Eukaryotic replication protein A (RPA) is a single-stranded DNA-binding protein with multiple functions in DNA replication, repair, and genetic recombination. RPA contains an evolutionarily conserved 4-cysteine-type zinc finger motif (X(3)CX(2-4)CX(12-15)CX(2)C) that has a potential role in regulation of DNA replication and repair (Dong, J., Park, J-S., and Lee, S-H. (1999) Biochem. J. 337, 311-317 and Lin, Y.-L., Shivji, M. K. K., Chen, C., Kolodner, R., Wood, R. D., and Dutta, A. (1998) J. Biol. Chem. 273, 1453-1461), even though the zinc finger itself is not essential for its DNA binding activity (Kim, D. K., Stigger, E., and Lee, S.-H. (1996) J. Biol. Chem. 271, 15124-15129). Here, we show that RPA single-stranded DNA (ssDNA) binding activity is regulated by reduction-oxidation (redox) through its zinc finger domain. RPA-ssDNA interaction was stimulated 10-fold by the reducing agent, dithiothreitol (DTT), whereas treatment of RPA with oxidizing agent, diazene dicarboxylic acid bis[N,N-dimethylamide] (diamide), significantly reduced this interaction. The effect of diamide was reversed by the addition of excess DTT, suggesting that RPA ssDNA binding activity is regulated by redox. Redox regulation of RPA-ssDNA interaction was more effective in the presence of 0.2 M NaCl or higher. Cellular redox factor, thioredoxin, was able to replace DTT in stimulation of RPA DNA binding activity, suggesting that redox protein may be involved in RPA modulation in vivo. In contrast to wild-type RPA, zinc finger mutant (cysteine to alanine mutation at amino acid 486) did not require DTT for its ssDNA binding activity and is not affected by redox. Together, these results suggest a novel function for a putative zinc finger in the regulation of RPA DNA binding activity through cellular redox.