PAGE 3 OF 6 Nucleic Acids Research, 2007, Vol. 35, No. 15 e100 Figure 1. ESI–FT ICR MS analysis of ssDNAs. (A) The raw mass spectra of 3, 7, 15, 21, 30 and 37-mer ssDNAs. The synthesized ssDNAs (30-hydroxylated) were suspended in the reaction buﬀer without protein and puriﬁed according to the protocol described in MATERIALS AND METHODS section. Labeled peak (697.460) is corresponding to 7-mer ssDNA having three protons removed ([M-3H]3–). Inset shows the isotopic cluster for [M-3H]3– and their sodium adduct ion peaks. (B) The deconvoluted mass spectra of the ssDNAs. The length, measured mass (Me), theoretical mass (Mt) and deviation between measured and theoretical mass are indicated at the top of each peak. the nucleotide content will cause diﬃculty in identiﬁcation of the fragments since various potential structures correspond to a single mass. Figure 2 shows the deconvoluted mass spectra of reaction products. The substrate and product dsDNAs are completely denatured and detected as ssDNAs. Indeed, any remaining dsDNAs would have complicated the interpretation of the results. An increased number of peaks corresponding to DNA reaction products were detected as the enzyme concentra- tion increased. Several peaks are more abundant than others. However, we cannot quantitatively interpret this in terms of selectivity because the nucleotide content and length seems to have an eﬀect on the ionization eﬃciency of oligonucleotides (data not shown). Over 90% of these peaks were accurately identiﬁed as 5–12-mer fragments. The major fragments identiﬁed by mass spectrometric analysis are listed in Figure 3. This result strongly suggests that the nuclease activity of CTD has no obvious sequence-speciﬁcity. All of mass spectrometric analyses were performed in triplicate. Figure 2. ESI–FT ICR MS analysis of reaction products. The substrate dsDNA was reacted with 0 (A), 100 (B) or 200 (C) nM CTD. The products were puriﬁed according to the protocol described in MATERIALS AND METHODS section. The deconvoluted spectra of reacted products of 50-phosphorylated 17-bp dsDNA were shown. For clarity, information concerning the length and measured mass of the major species are shown above the corresponding peaks. There were a few unidentiﬁed fragments that corre- sponded in mass to two or more candidate fragments (Figure 4A). Some of these peaks were subsequently identiﬁed by comparison with the result of a digestion of 50-hydroxylated dsDNA (Figure 4B). When the fragment contained an unreacted 50-terminus, the corresponding peak would shift as far as the diﬀerence between masses of hydroxyl and phosphoryl groups. As shown in Figure 3C, several peaks shifted and were identiﬁed. All of the theoretical masses used to identify the peaks were calculated by assuming the 50- and 30-termini of cleaved sites were phosphorylated and hydroxylated, respectively. These results indicate that CTD incised the phosphate backbone of oligodeoxynucleotides at the 30 side of the phosphates and the nicks generated by CTD could be ligated by DNA ligase. Thus, ESI–FT ICR MS can identify the nature of the cleaved sites with considerable accuracy. The nuclease activity for 17-bp dsDNA was also examined by electrophoretic analysis using a sequencing gel. As shown in Figure 6, the fragments were estimated to be 5–12-mers by comparison to the DNA size marker made by the Maxam–Gilbert method (21). These results are entirely consistent with the analysis by mass spectro- metry. Although a few fragments shorter or longer than 5- or 12-mer were observed when the enzyme concentra- tion was increased (data not shown), main products were always 5–12-mer fragments. This result indicates that a certain length of dsDNA is required for the formation of a stable CTD–substrate complex. All of the detected fragments retained an unreacted 50- or 30-terminus, indicating that CTD possesses a non- sequence-speciﬁc endonuclease activity rather than