The abortive synthesis of new DNA chains in Ehrlich ascites tumour cells treated with nitrogen mustard (HN2)

Nitrogen mustard (HN2)-sensitive Ehrlich ascites tumour cells, exposed to HN2 in vivo, showed an inhibition of DNA synthesis which increased with dosage. The effects of alkylation involved at least three distinct components: (1) interference with new 9S DNA chain formation, (2) slowing of the rate of chain growth and (3) loss of newly formed short chains. The dominant effect seemed to be abortive synthesis of new 9S chains; this effect could account for most of the inhibition of DNA synthesis if an initial rapid synthesis of 9S DNA were accompanied by an initial rapid rate of destruction of these chains. By relating the known frequencies of guaninyl alkylations to the postulated ‘replicon’ size observed in control experiments, it appears that only difunctional alkylation frequencies can be directly correlated with the inhibition of discontinuous DNA synthesis by HN2, Mechanistically, this could reflect interference of di-guaninyl alkylations with the integration of 9S chains into 30S, 44S and higher molecular weight species of DNA by ligases. The resulting obstructed short chains, ≦ 9S, might be exposed and so be vulnerable to destruction by the increased nuclease activities expected after alkylation.     

Crystal structure of the RNA demethylase ALKBH5 from zebrafish

ALKBH5, a member of AlkB family proteins, has been reported as a mammalian N⁶-methyladenosine (m⁶A) RNA demethylase. Here we report the crystal structure of zebrafish ALKBH5 (fALKBH5) with the resolution of 1.65 Å. Structural superimposition shows that fALKBH5 is comprised of a conserved jelly-roll motif. However, it possesses a loop that interferes potential binding of a duplex nucleic acid substrate, suggesting an important role in substrate selection. In addition, several active site residues are different between the two known m⁶A RNA demethylases, ALKBH5 and FTO, which may result in their slightly different pathways of m⁶A demethylation.     

Dodecameric structure and ATPase activity of the human TIP48/TIP49 complex

TIP48 and TIP49 are two related and highly conserved eukaryotic AAA(+) proteins with an essential biological function and a critical role in major pathways that are closely linked to cancer. They are found together as components of several highly conserved chromatin-modifying complexes. Both proteins show sequence homology to bacterial RuvB but the nature and mechanism of their biochemical role remain unknown. Recombinant human TIP48 and TIP49 were assembled into a stable high molecular mass equimolar complex and tested for activity in vitro. TIP48/TIP49 complex formation resulted in synergistic increase in ATPase activity but ATP hydrolysis was not stimulated in the presence of single-stranded, double-stranded or four-way junction DNA and no DNA helicase or branch migration activity could be detected. Complexes with catalytic defects in either TIP48 or TIP49 had no ATPase activity showing that both proteins within the TIP48/TIP49 complex are required for ATP hydrolysis. The structure of the TIP48/TIP49 complex was examined by negative stain electron microscopy. Three-dimensional reconstruction at 20 A resolution revealed that the TIP48/TIP49 complex consisted of two stacked hexameric rings with C6 symmetry. The top and bottom rings showed substantial structural differences. Interestingly, TIP48 formed oligomers in the presence of adenine nucleotides, whilst TIP49 did not. The results point to biochemical differences between TIP48 and TIP49, which may explain the structural differences between the two hexameric rings and could be significant for specialised functions that the proteins perform individually.     

The mRNA-binding site of annexin A2 resides in helices C-D of its domain IV

Annexin A2 (AnxA2) is a Ca(2+)-binding and phospholipid-binding protein involved in different intracellular processes including exocytosis, endocytosis and membrane-cytoskeleton movements. We have previously identified AnxA2 as an mRNA-binding protein present in cytoskeleton-bound polysomes, that binds to a specific approximately 100 nucleotide region in the 3'-untranslated region of c-myc and its cognate mRNA. In the present study, we show by UV cross-linking assays and surface plasmon resonance analyses that the mRNA-binding site of AnxA2 resides in its domain IV. Furthermore, the interaction of full-length AnxA2 with the 3'-untranslated region of anxA2 mRNA is Ca(2+)-dependent. By contrast, the interaction is Ca(2+)-independent for the isolated domain IV of AnxA2, suggesting that the mRNA-binding site is masked in Apo-AnxA2 and gains exposure through a Ca(2+)-induced conformational change of AnxA2 generating a favourable mRNA-binding site. The AnxA2-mRNA interaction is specific and involves helices C and D in domain IV of AnxA2, since point mutagenesis of several charged and polar exposed residues of these helices in the full-length protein strongly reduce RNA binding. The interaction appears to be sequential involving an initial phase of recognition dominated by electrostatic interactions, most likely between lysine residues and the phosphate backbone of RNA, followed by a second phase contributing to the specificity of the interaction.     

Increase in CpG DNA-induced inflammatory responses by DNA oxidation in macrophages and mice

Unmethylated CpG dinucleotide (CpG motif) is involved in the exacerbation of DNA-associated autoimmune diseases. We investigated the effect of DNA containing 8-hydroxydeoxyguanosine (oxo-dG), a representative DNA biomarker for oxidative stress in the diseases, on CpG motif-dependent inflammatory responses. ODN1668 and ODN1720 were selected as CpG-DNA and non-CpG DNA, respectively. Deoxyguanosine in the CpG motif (G9) or outside the motif (G15) of ODN1668 was substituted with oxo-dG to obtain oxo(G9)-1668 and oxo(G15)-1668, respectively. Oxo(G15)-1668 induced a significantly higher amount of tumor necrosis factor (TNF)-α from RAW264.7 macrophage-like cells than ODN1668, whereas oxo(G9)-1668, oxo(G8)-1720, or oxo(G15)-1720 hardly did. CpG DNA-induced TNF-α production was significantly increased by addition of oxo(G8)-1720 or oxo(G15)-1720, but not of ODN1720. This oxo-dG-containing DNA-induced increase in TNF-α production was also observed in primary cultured macrophages isolated from wild-type mice, but not observed in those from Toll-like receptor (TLR)-9 knockout mice. In addition, TNF-α production by ligands for TLR3, TLR4, or TLR7 was not affected by oxo-dG-containing DNA. Then, the footpad swelling induced by subcutaneous injection of ODN1668 into mice was increased by coinjection with oxo(G8)-1720, but not with ODN1720. These results indicate that oxo-dG-containing DNA increases the CpG motif-dependent inflammatory responses, which would exacerbate DNA-related autoimmune diseases.     

Mismatch repair factor MSH2-MSH3 binds and alters the conformation of branched DNA structures predicted to form during genetic recombination

Genetic studies in Saccharomyces cerevisiae predict that the mismatch repair (MMR) factor MSH2-MSH3 binds and stabilizes branched recombination intermediates that form during single strand annealing and gene conversion. To test this model, we constructed a series of DNA substrates that are predicted to form during these recombination events. We show in an electrophoretic mobility shift assay that S. cerevisiae MSH2-MSH3 specifically binds branched DNA substrates containing 3' single-stranded DNA and that ATP stimulates its release from these substrates. Chemical footprinting analyses indicate that MSH2-MSH3 specifically binds at the double-strand/single-strand junction of branched substrates, alters its conformation and opens up the junction. Therefore, MSH2-MSH3 binding to its substrates creates a unique nucleoprotein structure that may signal downstream steps in repair that include interactions with MMR and nucleotide excision repair factors.     

The p53-induced Wig-1 protein binds double-stranded RNAs with structural characteristics of siRNAs and miRNAs

Wig-1 is a p53-induced zinc finger protein. Here we show that human Wig-1 binds long (>or=23 bp) dsRNAs with 5'-overhangs. The first zinc finger domain is necessary but not sufficient for this dsRNA-binding in vitro. Wig-1 also binds dsRNA in living cells via zinc fingers 1 and 2. Both zinc fingers 1 and 2 are important for Wig-1-mediated growth suppression. Moreover, Wig-1 binds 21 bp dsRNAs with 3'-protruding ends. These findings demonstrate that human Wig-1 can bind different types of dsRNAs, including dsRNAs resembling small interfering RNAs (siRNAs) and microRNAs (miRNAs), and indicate that dsRNA binding has a role in Wig-1-mediated regulation of cell growth.     

RNA secondary structure mediates alternative 3'ss selection in Saccharomyces cerevisiae

Alternative splicing is the mechanism by which different combinations of exons in the pre-mRNA give rise to distinct mature mRNAs. This process is mediated by splicing factors that bind the pre-mRNA and affect the recognition of its splicing signals. Saccharomyces species lack many of the regulatory factors present in metazoans. Accordingly, it is generally assumed that the amount of alternative splicing is limited. However, there is recent compelling evidence that yeast have functional alternative splicing, mainly in response to environmental conditions. We have previously shown that sequence and structure properties of the pre-mRNA could explain the selection of 3' splice sites (ss) in Saccharomyces cerevisiae. In this work, we extend our previous observations to build a computational classifier that explains most of the annotated 3'ss in the CDS and 5' UTR of this organism. Moreover, we show that the same rules can explain the selection of alternative 3'ss. Experimental validation of a number of predicted alternative 3'ss shows that their usage is low compared to annotated 3'ss. The majority of these alternative 3'ss introduce premature termination codons (PTCs), suggesting a role in expression regulation. Furthermore, a genome-wide analysis of the effect of temperature, followed by experimental validation, yields only a small number of changes, indicating that this type of regulation is not widespread. Our results are consistent with the presence of alternative 3'ss selection in yeast mediated by the pre-mRNA structure, which can be responsive to external cues, like temperature, and is possibly related to the control of gene expression.     

Influence of DNA end structure on the mechanism of initiation of DNA unwinding by the Escherichia coli RecBCD and RecBC helicases

Escherichiacoli RecBCD is a bipolar DNA helicase possessing two motor subunits (RecB, a 3′-to-5′ translocase, and RecD, a 5′-to-3′ translocase) that is involved in the major pathway of recombinational repair. Previous studies indicated that the minimal kinetic mechanism needed to describe the ATP-dependent unwinding of blunt-ended DNA by RecBCD in vitro is a sequential n-step mechanism with two to three additional kinetic steps prior to initiating DNA unwinding. Since RecBCD can “melt out” ∼ 6 bp upon binding to the end of a blunt-ended DNA duplex in a Mg²⁺-dependent but ATP-independent reaction, we investigated the effects of noncomplementary single-stranded (ss) DNA tails [3′-(dT)₆ and 5′-(dT)₆ or 5′-(dT)₁₀] on the mechanism of RecBCD and RecBC unwinding of duplex DNA using rapid kinetic methods. As with blunt-ended DNA, RecBCD unwinding of DNA possessing 3′-(dT)₆ and 5′-(dT)₆ noncomplementary ssDNA tails is well described by a sequential n-step mechanism with the same unwinding rate (mk_U = 774 ± 16 bp s^− 1) and kinetic step size (m = 3.3 ± 1.3 bp), yet two to three additional kinetic steps are still required prior to initiation of DNA unwinding (k_C = 45 ± 2 s^− 1). However, when the noncomplementary 5′ ssDNA tail is extended to 10 nt [5′-(dT)₁₀ and 3′-(dT)₆], the DNA end structure for which RecBCD displays optimal binding affinity, the additional kinetic steps are no longer needed, although a slightly slower unwinding rate (mk_U = 538 ± 24 bp s^− 1) is observed with a similar kinetic step size (m = 3.9 ± 0.5 bp). The RecBC DNA helicase (without the RecD subunit) does not initiate unwinding efficiently from a blunt DNA end. However, RecBC does initiate well from a DNA end possessing noncomplementary twin 5′-(dT)₆ and 3′-(dT)₆ tails, and unwinding can be described by a simple uniform n-step sequential scheme, without the need for the additional k_C initiation steps, with a similar kinetic step size (m = 4.4 ± 1.7 bp) and unwinding rate (mk_obs = 396 ± 15 bp s^− 1). These results suggest that the additional kinetic steps with rate constant k_C required for RecBCD to initiate unwinding of blunt-ended and twin (dT)₆-tailed DNA reflect processes needed to engage the RecD motor with the 5′ ssDNA.