From the Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom
Received for publication, March 13, 2003
, and in revised form, April 4, 2003.
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ABSTRACT |
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INTRODUCTION |
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Recent work has now provided strong genetic evidence that deamination of cytosine can also occur in DNA as part of a physiological program. Thus, it appears that deamination of dC dU within the immunoglobulin loci is triggered by AID1 (activation-induced deaminase), a B lymphocyte-specific member of the APOBEC family, where it initiates major pathways of antibody gene diversification (7, 8, 9, 10, 11, 12). Furthermore, several members of the APOBEC family (including APOBEC1 itself) when expressed in bacteria appear to cause mutation through deamination of cytosine in DNA (10, 13). Quite apart from the homology of the APOBEC proteins to bacterial cytidine deaminase (4), the genetic evidence very strongly indicates that the APOBEC family members themselves are intimately involved in the DNA deamination reaction. Thus, the mutations generated by APOBEC family members are all transitions at dC:dG with the frequency and nature of the nucleotide substitutions affected by deficiency in uracil-DNA glycosylase. Furthermore, the local sequence context of the dC residue targeted for mutation differs between APOBEC family members (10, 13). Nevertheless, there is a need for biochemical evidence that APOBEC family members can trigger such deamination in vitro, not merely to confirm the predictions of the genetic analyses but also so as to facilitate investigation into the kinetics, substrate specificity, and characteristics of the deamination reaction. Here, we describe an important step in that direction and show that the substrate for the deamination is single-stranded DNA and affected by local sequence context.
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EXPERIMENTAL PROCEDURES |
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OligodeoxyribonucleotidesThe oligodeoxyribonucleotides used are listed in Table I.
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Preparation of Recombinant APOBEC1A 2-ml overnight culture of a fresh E. coli transformant grown in LB, 0.2% glucose, 50 µg/ml carbenicillin was diluted into 300 ml of the same medium and grown at 37 °C to an A600 of 0.8. The culture was chilled on ice for 20 min and then incubated with aeration for 16 h at 16 °C in the presence of inducer (1 mM isopropyl-1-thio--D-galactopyranoside). Cells were harvested by centrifugation, washed, and resuspended in 20 ml H buffer (50 mM Tris, pH 7.4, 50 mM KCl, 5 mM EDTA, 1 mM DTT, and a protease inhibitor mixture (Roche Diagnostics)). Following sonication and ultracentrifugation (100,000 x g for 45 min), the supernatant was passed through a 0.2-µm filter and applied to a Sepharose fast-flow Mono Q column (Amersham Biosciences; 10 ml bed volume). After washing with 7 column volumes of buffer H, bound proteins were eluted in buffer H supplemented with increasing salt concentrations (from 50 to 1500 mM Cl-) collecting 15-ml fractions. Fractions and flow-through were concentrated 100-fold using VivaSpin concentrators (Mr 10,000 cut-off) (VivaScience) and assayed. Samples eluting with 10001500 mM salt were pooled and loaded in a volume of 0.5 ml onto a HighPrep Sephacryl S-200 high resolution 16/60 gel-filtration column (Amersham Biosciences) in buffer H. Fractions (1 ml) were collected and concentrated 20-fold before analysis.
TLC-based Deaminase AssaySamples (24 µl) were incubated at 37 °C for 5 h in 20 µl of buffer R (40 mM Tris, pH 8, 40 mM KCl, 50 mM NaCl, 5 mM EDTA, 1 mM DTT, 10% glycerol) containing 75,000 cpm of -32P-dC-labeled single-stranded DNA (prepared by a 3 min heating to 95 °C of the products of asymmetric PCR amplification of the lacI region in pTrc99 performed using [
-32P]dCTP (3000 Ci/mmol)). Following phenol extraction and ethanol precipitation, the DNA was digested with Penicillium citrinum P1 nuclease (Sigma) overnight at 37 °C (14), and the P1 digests then subjected to thin layer chromatography on polyethyleneimine-cellulose in either (i) 0.5 M LiCl at 4 °C or (ii) at room temperature in 1 M CH3COOH until the buffer front had migrated 2.5 cm and then in 0.9 M CH3COOH:0.3 M LiCl (15). Products were detected using a phosphorimager. Chemical deamination of cytosine in DNA using bisulfite/hydroquinone was performed as described previously (16).
UDG-based Deaminase AssaySamples (12 µl) were incubated at 37 °C for 2 h in 10 µl of buffer R with 5'-biotinylated oligonucleotides that either were synthesized with fluorescein at their 3'-ends (3 pmol of oligonucleotide per reaction) or were 3'-labeled by ligation with [-32P]dideoxyadenylate (100,000 cpm; 0.1 pmol) using terminal deoxynucleotidyltransferase. Reactions were terminated by heating to 90 °C for 3 min and oligonucleotides purified on streptavidin magnetic beads (Dynal), washing at 72 °C (except in Fig. 2A, where the streptavidin purification step was omitted). Deamination of cytosine in the oligonucleotides was monitored by incubating the bead-immobilized oligonucleotides at 37 °C for 30 min with excess uracil-DNA glycosylase (0.5 unit of UDG; enzyme and buffer from New England Biolabs) and then bringing the sample to 0.15 M in NaOH and incubating for a further 30 min. The oligonucleotides were then subjected to electrophoresis on 15% PAGE-urea gels which were developed by either fluorescence detection or phosphorimager analysis.
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Western BlottingWestern blot detection of APOBEC1 following SDS-PAGE of samples that had been diluted 20100-fold was performed using a goat-anti-APOBEC1 serum (Santa Cruz Biotechnology), developing with horseradish peroxidase-conjugated donkey anti-goat immunoglobulin antiserum (Binding Site, Birmingham, UK). Low range molecular weight markers were from Bio-Rad.
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RESULTS |
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We considered the possibility that the DNA deaminating activity might be specific for single-stranded substrates but that this activity might be masked by nonspecific nucleases. We therefore devised an assay that would be less sensitive to contaminating nucleases (Fig. 1A). The bacterial extracts were incubated with -32P-dC-labeled single-stranded DNA which was then purified, digested with nuclease P1, and subjected to thin layer chromatography to test for the presence of [
-32P]dUMP. Clear evidence of dC deamination in this assay was detected using extracts of E. coli expressing two different APOBEC1 constructs but not from control extracts or from extracts made from E. coli cells carrying plasmids expressing mutant APOBEC1, APOBEC2, or dCTP deaminase (none of which function as DNA mutators in the bacterial assay (13)) (Fig. 1B). The DNA deaminase activity was also evident in APOBEC1 transformants of a mutant E. coli deficient in both dcd- and cdd-encoded deaminases (Fig. 1B, iii). That the product of APOBEC1 action was indeed dUMP is indicated by the co-migration of the radioactive product with dUMP in two distinct buffer systems. These results encouraged us to fractionate the extracts of APOBEC1-transformed E. coli to see whether the DNA deamination activity could be sufficiently separated from nonspecific nucleases so as to be detectable using the oligonucleotide cleavage assay.
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Partial PurificationPilot experiments revealed that ion-exchange chromatography could be used to obtain samples of APOBEC1 that contained diminished nonspecific nuclease activity. Thus, while only a proportion of the APOBEC1 polypeptide bound to the Mono Q column (around 1020% based on ECL quantitation of the Western blot assay), elution of this bound fraction with >0.8 M Cl- yielded a sample that displayed cytosine-DNA deamination activity (as monitored using the TLC-based assay) but containing diminished nonspecific nuclease activity in the UDG-based assay (Fig. 2A). These fractions were then concentrated and subjected to gel filtration (Fig. 2B). The major APOBEC1 peak eluted in fractions 79 (corresponding to an Mr of 95140,000) co-eluting with peak DNA deaminating activity. Indeed, with these fractions from the gel filtration column, DNA deamination could now readily be detected by the UDG-based assay using a single-stranded oligonucleotide substrate (although the peak fractions also contained activity that removed the 3'-label from the oligonucleotide). Mass spectrometric analysis of proteins in fraction 9 following SDS-PAGE revealed the recombinant APOBEC1 migrating at the position marked by the asterisked in Fig. 2B, i, although the majority of the bands derived from ribosomal proteins.
Characteristics of the DNA Deaminating ActivityThe UDG-based deaminase assay was used to monitor the specificity and characteristics of the partially purified APOBEC1 (Fig. 3A). Samples were incubated with a single-stranded oligodeoxyribonucleotide (with or without its complement), which contained internal dC residue(s) and that was 5'-biotinylated as well as 3'-labeled. After purification on streptavidin, the oligonucleotide was treated with UDG (plus alkali), resulting in site-specific cleavage if the oligonucleotide had been subjected to dC dU deamination. Thus, deamination is read out by the appearance of the specific cleavage product following PAGE-urea analysis.
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The partially purified wild type protein (but not the Glu63 Ala mutant) showed clear activity on a single-stranded oligonucleotide with the cleavage being dependent on the subsequent incubation with UDG (Fig. 3, B and C). The deaminating activity was not inhibited by tetrahydrouridine (which inhibits cytidine deaminases (17)) or by RNase (Fig. 3D). Strikingly (and consistent with our inability to detect deamination on double-stranded oligonucleotide substrates using crude extracts of bacterial transformants (see above)), the activity was blocked if a complementary (but not if an irrelevant) oligonucleotide was titrated into the assay (Fig. 3E). Examination of the cleavage products generated in the UDG-based assay suggests that not all dC residues are equally susceptible to APOBEC1-mediated deamination. While the data do not allow us yet to identify the ideal in vitro target for APOBEC1-mediated deamination, it is clear that in oligonucleotide SPM168 the third cytosine in the sequence TCCGCG is much less favored than the other two (Fig. 3, BE). Similarly, evidence of specificity comes from comparing various related oligonucleotides as substrates, where all the data taken together point to deamination being especially disfavored when a purine is located immediately 5' of the cytosine (Fig. 3, F and G).
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DISCUSSION |
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While these results now provide biochemical evidence of DNA deamination in vitro by an APOBEC family member, the DNA deaminating activity that we see here is weak. Thus, given the abundance of APOBEC1 polypeptide in the peak fraction from the gel filtration column, it appears that, on average, each molecule of recombinant APOBEC1 is responsible for in the order of a single deamination event in a 10-min incubation in the UDG-based assay. The weakness of this activity could of course reflect that the assay is suboptimal and might be enhanced by additional components or that the recombinant APOBEC1 is improperly folded, lacking some post-translational modification or unstable. However, crude calculations indicate that if the 500 molecules of APOBEC1 expressed in each E. coli transformant displayed a DNA deamination activity of this order in vivo and if this were targeted randomly to all cytosine residues in the genome, then this could, in principle, be more than sufficient to account for the several 1000-fold enhanced mutation frequencies seen at the rpoB and other loci in UDG-deficient E. coli following 20 generations of growth (13). Similarly, somatic hypermutation of immunoglobulin variable genes by targeted AID-mediated dC deamination may involve a single and most probably less than 10 targeted dC deamination events in each B lymphocyte cell cycle.
The results reveal a clear sensitivity to the local sequence context of the APOBEC1-mediated dC deamination in the in vitro assay, most readily explained by a bias against a 5'-flanking purine residue. This would accord well with the in vivo data where a near-total restriction to mutation at dC residues with a 5'-flanking pyrimidine is seen at the rpoB locus (13) as well as a similar bias at another test locus in E. coli.2 Curiously, however, these findings contrast with the fact that the target cytosine of APOBEC1 in apolipoprotein B RNA is flanked by a 5'-A residue (2, 3). It will obviously be interesting to obtain more information about both the structural and kinetic features of the interaction and activity of recombinant APOBEC1 on different RNA and DNA substrates.
The in vitro assay also reveals that APOBEC1 deamination is targeted to single-stranded DNA and, indeed, was undetectable on double-stranded DNA. This specificity for single-stranded DNA is in accordance with the fact that the natural substrate of APOBEC1 is most likely single-stranded RNA (5), and presumably, the same active site in APOBEC1 is used for both types of polynucleotide. Furthermore, spontaneous deamination of cytosine is also much more rapid in single- (as opposed to double-) stranded DNA (19). If this in vitro preference of APOBEC1 for a single-stranded DNA substrate can be extrapolated to the in vivo situation as well as to other DNA-mutating APOBEC family members, it could go some way to explaining why AID-mediated DNA changes (somatic hypermutation and class switch recombination) correlate with transcription of the DNA target gene. Clearly, extending on the assays described here and applying them to other APOBEC family members should give valuable insight into the mechanism and specificity of APOBEC family-mediated DNA deamination.
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FOOTNOTES |
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To whom correspondence may be addressed. Tel.: 44-1223-402269; Fax: 44-1223-412178; E-mail: skpm{at}mrc-lmb.cam.ac.uk (for S. K. P.-M) or msn{at}mrc-lmb.cam.ac.uk (for M. S. N.).
1 The abbreviations used are: AID, activation-induced deaminase; APOBEC1, apolipoprotein B-editing complex catalytic subunit 1; UDG, uracil-DNA glycosylase; DTT, dithiothreitol; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight.
2 R. C. L. Beale and M. S. Neuberger, unpublished data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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