Single-stranded DNA scanning and deamination by APOBEC3G cytidine deaminase at single molecule resolution

APOBEC3G (Apo3G) is a single-stranded (ss)DNA cytosine deaminase that eliminates HIV-1 infectivity by converting C → U in numerous small target motifs on the minus viral cDNA. Apo3G deaminates linear ssDNA in vitro with pronounced spatial asymmetry favoring the 3′ → 5′ direction. A similar polarity observed in vivo is believed responsible for initiating localized C → T mutational gradients that inactivate the virus. When compared with double-stranded (ds)DNA scanning enzymes, e.g. DNA glycosylases that excise rare aberrant bases, there is a paucity of mechanistic studies on ssDNA scanning enzymes. Here, we investigate ssDNA scanning and motif-targeting mechanisms for Apo3G using single molecule Förster resonance energy transfer. We address the specific issue of deamination asymmetry within the general context of ssDNA scanning mechanisms and show that Apo3G scanning trajectories, ssDNA contraction, and deamination efficiencies depend on motif sequence, location, and ionic strength. Notably, we observe the presence of bidirectional quasi-localized scanning of Apo3G occurring proximal to a 5′ hot motif, a motif-dependent DNA contraction greatest for 5′ hot > 3′ hot > 5′ cold motifs, and diminished mobility at low salt. We discuss the single molecule Förster resonance energy transfer data in terms of a model in which deamination polarity occurs as a consequence of Apo3G binding to ssDNA in two orientations, one that is catalytically favorable, with the other disfavorable.     

Single-stranded structure of newly replicated DNA during lens regeneration.

Nascent (newly synthesized) DNA obtained from the regenerating len-system of Triturus is first isolated as single stranded molecules. These imtermediates are later converted into double-stranded molecules.     

Differences in the enzymatic efficiency of human and bony fish AID are mediated by a single residue in the C terminus modulating single-stranded DNA binding

Activation-induced cytidine deaminase (AID) mediates antibody diversification by deaminating deoxycytidines to deoxyuridine within immunoglobulin genes. However, it also generates genome-wide DNA lesions, leading to transformation. Though the biochemical properties of AID have been described, its 3-dimensional structure has not been determined. Hence, to investigate the relationship between the primary structure and biochemical characteristics of AID, we compared the properties of human and bony fish AID, since these are most divergent in amino acid sequence. We show that AIDs of various species have different catalytic rates that are thermosensitive and optimal at native physiological temperatures. Zebrafish AID is severalfold more catalytically robust than human AID, while catfish AID is least active. This disparity is mediated by a single amino acid difference in the C terminus. Using functional assays supported by models of AID core and surface structure, we show that this residue modulates activity by affecting ssDNA binding. Furthermore, the cold-adapted catalytic rates of fish AID result from increased ssDNA binding affinity at lower temperatures. Our work suggests that AID may generate DNA damage with variable efficiencies in different organisms, identifies residues critical in regulating AID activity, and provides insights into the evolution of the APOBEC family of enzymes.     

Single-stranded DNA structure and DNA polymerase activity in the presence of nucleic acid helix-unwinding proteins from calf thymus.

In the preceding articles we have described the isolation and some of the properties of two calf thymus proteins which bind selectively to single-stranded DNA and which appear analogous to previously isolated prokaryotic DNA-unwinding proteins. In the present work we demonstrate two further points of analogy. First, both the calf UP1 and the high salt eluting proteins form protein-rich complexes with single-stranded DNA, and hold this DNA in a rigid, extended conformation. Second, these proteins stimulate the calf thymus DNA polymerase-alpha; phage T4 gene 32-protein does not. The stimulation of a homologous DNA polymerase is characteristic of several prokaryotic DNA-unwinding proteins and is assumed to reflect their in vivo role in DNA synthesis.     

Effect of target secondary structure on RNAi efficiency

RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) has become a powerful tool for gene knockdown studies. However, the levels of knockdown vary greatly. Here, we examine the effect of target disruption energy, a novel measure of target accessibility, along with other parameters that may affect RNAi efficiency. Based on target secondary structures predicted by the Sfold program, the target disruption energy represents the free energy cost for local alteration of the target structure to allow target binding by the siRNA guide strand. In analyses of 100 siRNAs and 101 shRNAs targeted to 103 endogenous human genes, we find that the disruption energy is an important determinant of RNAi activity and the asymmetry of siRNA duplex asymmetry is important for facilitating the assembly of the RNA-induced silencing complex (RISC). We estimate that target accessibility and duplex asymmetry can improve the target knockdown level significantly by nearly 40% and 26%, respectively. In the RNAi pathway, RISC assembly precedes target binding by the siRNA guide strand. Thus, our findings suggest that duplex asymmetry has significant upstream effect on RISC assembly and target accessibility has strong downstream effect on target recognition. The results of the analyses suggest criteria for improving the design of siRNAs and shRNAs.     

Evolution of the AID/APOBEC family of polynucleotide (deoxy)cytidine deaminases

The AID/APOBEC family (comprising AID, APOBEC1, APOBEC2, and APOBEC3 subgroups) contains members that can deaminate cytidine in RNA and/or DNA and exhibit diverse physiological functions (AID and APOBEC3 deaminating DNA to trigger pathways in adaptive and innate immunity; APOBEC1 mediating apolipoprotein B RNA editing). The founder member APOBEC1, which has been used as a paradigm, is an RNA-editing enzyme with proposed antecedents in yeast. Here, we have undertaken phylogenetic analysis to glean insight into the primary physiological function of the AID/APOBEC family. We find that although the family forms part of a larger superfamily of deaminases distributed throughout the biological world, the AID/APOBEC family itself is restricted to vertebrates with homologs of AID (a DNA deaminase that triggers antibody gene diversification) and of APOBEC2 (unknown function) identifiable in sequence databases from bony fish, birds, amphibians, and mammals. The cloning of an AID homolog from dogfish reveals that AID extends at least as far back as cartilaginous fish. Like mammalian AID, the pufferfish AID homolog can trigger deoxycytidine deamination in DNA but, consistent with its cold-blooded origin, is thermolabile. The fine specificity of its mutator activity and the biased codon usage in pufferfish IgV genes appear broadly similar to that of their mammalian counterparts, consistent with a coevolution of the antibody mutator and its substrate for the optimal targeting of somatic mutation during antibody maturation. By contrast, APOBEC1 and APOBEC3 are later evolutionary arrivals with orthologs not found in pufferfish (although synteny with mammals is maintained in respect of the flanking loci). We conclude that AID and APOBEC2 are likely to be the ancestral members of the AID/APOBEC family (going back to the beginning of vertebrate speciation) with both APOBEC1 and APOBEC3 being mammal-specific derivatives of AID and a complex set of domain shuffling underpinning the expansion and evolution of the primate APOBEC3s.     

Regulation of Single-stranded DNA Binding by the C Termini of Escherichia coli Single-stranded DNA-binding (SSB) Protein

The homotetrameric Escherichia coli single-stranded DNA-binding (SSB) protein plays a central role in DNA replication, repair, and recombination. In addition to its essential activity of binding to transiently formed single-stranded (ss) DNA, SSB also binds an array of partner proteins and recruits them to their sites of action using its four intrinsically disordered C-terminal tails. Here we show that the binding of ssDNA to SSB is inhibited by the SSB C-terminal tails, specifically by the last 8 highly acidic amino acids that comprise the binding site for its multiple partner proteins. We examined the energetics of ssDNA binding to short oligodeoxynucleotides and find that at moderate salt concentration, removal of the acidic C-terminal ends increases the intrinsic affinity for ssDNA and enhances the negative cooperativity between ssDNA binding sites, indicating that the C termini exert an inhibitory effect on ssDNA binding. This inhibitory effect decreases as the salt concentration increases. Binding of ssDNA to approximately half of the SSB subunits relieves the inhibitory effect for all of the subunits. The inhibition by the C termini is due primarily to a less favorable entropy change upon ssDNA binding. These observations explain why ssDNA binding to SSB enhances the affinity of SSB for its partner proteins and suggest that the C termini of SSB may interact, at least transiently, with its ssDNA binding sites. This inhibition and its relief by ssDNA binding suggest a mechanism that enhances the ability of SSB to selectively recruit its partner proteins to sites on DNA.     

Single-stranded DNA binding proteins unwind the newly synthesized double-stranded DNA of model miniforks

Single-stranded DNA binding (SSB) proteins are essential proteins of DNA metabolism. We characterized the binding of the bacteriophage T4 SSB, Escherichia coli SSB, human replication protein A (hRPA), and human hSSB1 proteins onto model miniforks and double-stranded-single-stranded (ds-ss) junctions exposing 3' or 5' ssDNA overhangs. T4 SSB proteins, E. coli SSB proteins, and hRPA have a different binding preference for the ss tail exposed on model miniforks and ds-ss junctions. The T4 SSB protein preferentially binds substrates with 5' ss tails, whereas the E. coli SSB protein and hRPA show a preference for substrates with 3' ss overhangs. When interacting with ds-ss junctions or miniforks, the T4 SSB protein, E. coli SSB protein, and hRPA can destabilize not only the ds part of a ds-ss junction but also the daughter ds arm of a minifork. The T4 SSB protein displays these unwinding activities in a polar manner. Taken together, our results position the SSB protein as a potential key player in the reversal of a stalled replication fork and in gap repair-mediated repetitive sequence expansion.     

Probing the efficiency of proteolytic events by positional proteomics

Several mass spectrometry-driven techniques allow to map the substrate repertoires and specificities of proteases. These techniques typically yield long lists of protease substrates and processed sites with (potential) physiological relevance, but in order to understand the primary function of a protease, it is important to discern bystander substrates from critical substrates. Because the former are generally processed with lower efficiency, data on the actual substrate cleavage efficiency could assist in categorizing protease substrates. In this study, quantitative mass spectrometry following metabolic proteome labeling (SILAC), combined with the isolation of N-terminal peptides by Combined Fractional Diagonal Chromatography, was used to monitor fluxes in the concentration of protease-generated neo-N-termini. In our experimental setup, a Jurkat cell lysate was treated with the human serine protease granzyme B (hGrB) for three different incubation periods. The extensive list of human granzyme B substrates previously catalogued by N-terminal Combined Fractional Diagonal Chromatography (1) was then used to assign 101 unique hGrB-specific neo-N-termini in 86 proteins. In this way, we were able to define several sites as getting efficiently cleaved in vitro and consequently recognize potential physiologically more relevant substrates. Among them the well-known hGrB substrate Bid was confirmed as being an efficient hGrB substrate next to several other potential regulators of hGrB induced apoptosis such as Bnip2 and Akap-8. Several of our proteomics results were further confirmed by substrate immunoblotting and by using peptide substrates incubated with human granzyme B.     

Isolation, tissue distribution, and chromosomal localization of the human activation-induced cytidine deaminase (AID) gene

The gene encoding activation-induced cytidine deaminase (AID), a member of the cytidine deaminase family, was isolated from a murine B cell lymphoma line, CH12F3-2, induced by combined stimulation of TGF-beta, IL-4, and CD40L. We have isolated the human orthologue of mouse AID cDNA, which has an open reading frame of 198 residues containing a conserved cytidine deaminase motif. The amino acid sequence of human AID is 92% identical to that of mouse AID. RT-PCR analysis of 15 human tissues showed that AID mRNA is expressed strongly in lymph nodes and tonsils. The complete human AID gene consisting of five exons was isolated and mapped to chromosome 12p13 by fluorescence in situ hybridization.     

Ubiquitination and filamentous structure of cytidine triphosphate synthase

Living organisms respond to nutrient availability by regulating the activity of metabolic enzymes. Therefore, the reversible post-translational modification of an enzyme is a common regulatory mechanism for energy conservation. Recently, cytidine-5′-triphosphate (CTP) synthase was discovered to form a filamentous structure that is evolutionarily conserved from flies to humans. Interestingly, induction of the formation of CTP synthase filament is responsive to starvation or glutamine depletion. However, the biological roles of this structure remain elusive. We have recently shown that ubiquitination regulates CTP synthase activity by promoting filament formation in Drosophila ovaries during endocycles. Intriguingly, although the ubiquitination process was required for filament formation induced by glutamine depletion, CTP synthase ubiquitination was found to be inversely correlated with filament formation in Drosophila and human cell lines. In this article, we discuss the putative dual roles of ubiquitination, as well as its physiological implications, in the regulation of CTP synthase structure.     

15N kinetic isotope effects on uncatalyzed and enzymatic deamination of cytidine.

15N isotope effects and solvent deuterium isotope effects have been measured for the hydrolytic deamination of cytidine catalyzed by Escherichia coli cytidine deaminase and for the uncatalyzed reaction proceeding spontaneously in neutral solution at elevated temperatures. The primary (15)(V/K) arising from the exocyclic amino group for wild-type cytidine deaminase acting on its natural substrate, cytidine, is 1.0109 (in H(2)O, pH 7.3), 1.0123 (in H(2)O, pH 4.2), and 1.0086 (in D(2)O, pD 7.3). Increasing solvent D(2)O content has no substantial effect on k(cat) but enhances k(cat)/K(m), with a proton inventory showing that the fractionation factors of at least two protons increase markedly during the reaction. Mutant cytidine deaminases with reduced catalytic activity show more pronounced (15)N isotope effects of 1.0124 (Glu91Ala), 1.0134 (His102Ala), and 1.0158 (His102Asn) at pH 7.3 in H(2)O, as expected for processes in which the chemical transformation of the substrate becomes more rate determining. The isotope effect of mutant His102Asn is 1.033 after correcting for protonation of the -NH(2) group, and represents the intrinsic isotope effect on C-N bond cleavage. This result allows an estimation of the forward commitment of the reaction with the wild-type enzyme. The observed (15)N kinetic isotope effect of the pyrimidine N-3, for wild-type cytidine deaminase acting on cytidine, is 0.9879, which is consistent with protonation and rehybidization of N-3 with hydroxide ion attack on the adjacent carbon to create a tetrahedral intermediate. These results show that enzymatic deamination of cytidine proceeds stepwise through a tetrahedral intermediate with ammonia elimination as the major rate-determining step. The primary (15)N isotope effects observed for the uncatalyzed reaction at pH 7 (1.0021) and pH 12.5 (1.0034) were found to be insensitive to changing temperatures between 100 and 185 degrees C. These results show that the uncatalyzed and the enzymatic deaminations of cytidine proceed by similar mechanisms, although the commitment to C-N bond breaking is greater for the spontaneous reaction.     

Single-stranded DNA binding protein and DNA helicase of bacteriophage T7 mediate homologous DNA strand exchange.

Two proteins encoded by bacteriophage T7, the gene 2.5 single-stranded DNA binding protein and the gene 4 helicase, mediate homologous DNA strand exchange. Gene 2.5 protein stimulates homologous base pairing of two DNA molecules containing complementary single-stranded regions. The formation of a joint molecule consisting of circular, single-stranded M13 DNA, annealed to homologous linear, duplex DNA having 3'- or 5'-single-stranded termini of approximately 100 nucleotides requires stoichiometric amounts of gene 2.5 protein. In the presence of gene 4 helicase, strand transfer proceeds at a rate of > 120 nucleotides/s in a polar 5' to 3' direction with respect to the invading strand, resulting in the production of circular duplex M13 DNA. Strand transfer is coupled to the hydrolysis of a nucleoside 5'-triphosphate. The reaction is dependent on specific interactions between gene 2.5 protein and gene 4 protein.     

Probing the structure of multi-stranded guanine-rich DNA complexes by Raman spectroscopy and enzymatic degradation.

The multi-stranded DNA complexes formed by the oligonucleotides d(T15G4T2G4), Tel, and d(T15G15), TG, were examined by nuclease digestion and Raman spectroscopy. Both Tel and TG can aggregate to form structures consisting of multiple, parallel-oriented DNA strands with two independent structural domains. Overall, the structures of the TG and Tel aggregates appear similar. According to the Raman data, the majority of bases are in C2'-endo/anti conformation. The interaction of guanines at the 3'-ends in both complexes stabilizes the complexes and protects them from degradation by exonuclease III. The 5'-extensions remain single-stranded and the thymines are accessible to single-strand-specific nuclease digestion. The extent of enzymatic cleavage at the junction at the 5' end of the 15 thymines implies a conformational change between this part of the molecule and the guanine-rich region. The differential enzymatic sensitivity of the complexes suggests there are variations in backbone conformations between TG and Tel aggregates. TG aggregates were more resistant to digestion by DNase I, Mung Bean nuclease, and S1 nuclease than Tel complexes. It is proposed that the lower DNase I sensitivity may be partly due to the more stable backbone exhibited by TG than Tel complexes. Structural uniformity along the guanine core of TG is suggested, as there is no indication of structural discontinuities or protected sites in the guanine-rich regions of TG aggregates. The lower extent of digestion by Mung Bean nuclease at the 3' end implies that these bases are inaccessible to the enzyme. This suggests that there is minimal fraying at the ends, which is consistent with the extreme thermal stability of the TG aggregates.     

Sequence context effect on the structure of nitrous acid induced DNA interstrand cross-links

In the preceding paper in this journal, we described the solution structure of the nitrous acid cross-linked dodecamer duplex [d(GCATCCGGATGC)]2 (the cross-linked guanines are underlined). The structure revealed that the cross-linked guanines form a nearly planar covalently linked 'G:G base pair', with the complementary partner cytidines flipped out of the helix. Here we explore the flanking sequence context effect on the structure of nitrous acid cross-links in [d(CG)]2 and the factors allowing the extrahelical cytidines to adopt such fixed positions in the minor groove. We have used NMR spectroscopy to determine the solution structure of a second cross-linked dodecamer duplex, [d(CGCTACGTAGCG)]2, which shows that the identity of the flanking base pairs significantly alters the stacking patterns and phosphate backbone conformations. The cross-linked guanines are now stacked well on adenines preceding the extrahelical cytidines, illustrating the importance of purine- purine base stacking. Observation of an imino proton resonance at 15.6 p.p.m. provides evidence for hydrogen bonding between the two cross-linked guanines. Preliminary structural studies on the cross-linked duplex [d(CGCGACGTCGCG)]2 show that the extrahelical cytidines are very mobile in this sequence context. We suggest that favorable van der Waals interactions between the cytidine and the adenine 2 bp away from the cross-link localize the cytidines in the previous cross-linked structures.