To determine the roles of specific cluster properties on Ape1 cleavage, we required a substantial battery of oligonucleotides containing clusters with different numbers of lesions in specific orientations and polarities. shows the configurations of the complete set of oligonucleotides. Rather than synthesize each strand individually, we used a constant A1 strand containing one uracil and constructed strand B from two constant terminal oligonucleotides and a central variable section. -I shows the scheme for producing the dually labeled 51-bp duplex containing a complex bistranded clustered damage. Strand B consists of the two terminal oligonucleotides, Ba and Bc, where either or both can be labeled with a fluorophore. The 21mer central cassette Bb can contain one or more uracil residues. The components are annealed, ligated and the uracil residues converted to abasic sites by uracil-DNA glycosylase (UDG). We found that UDG effectively converted all uracil sites in the clusters shown in Table I to abasic sites (data not shown).
-II shows a true color gel with intact 51mer A1•B−5 (Lanes 1, 3 and 5) and their Ape1 cleavage products (Lanes 2, 4 and 6). -II shows the configuration of these oligonucleotides. In Lanes 1 and 2, strand A is 5′ labeled with 6-FAM, and strand B is 3′ labeled with TAMRA. In Lanes 3 and 4, strand A is unlabeled and strand B is labeled at its 3′ end with TAMRA and at its 5′ end with 6-FAM. Lane 5 and 6 contain unlabeled A1, and B dually labeled: 3′ with TAMRA and 5′ with JOE (6-carboxy-4′, 5′-dichloro-2′, 7′-dimethoxyfluorescein, light green). The image shows that fully, partially and unligated fragments, as well as cleavage products, can be readily identified from their fluorophore tags and migrations.
For quantitative analysis of cluster cleavage, we used a charged coupled device-based camera, in which external filters can be used to image selectively the emission of the fluorophore. We determined the direct proportionality between TAMRA and 6-FAM fluorescence and DNA amount (verified by quantifying the fluorescence of ethidium bromide staining—proportional to DNA mass—of the same bands). The fluorescence of JOE and TAMRA overlapped significantly, and therefore JOE was not used in dual-label experiments with TAMRA. Panel I of shows that the fluorescence—whether detected by filters that detect preferentially TAMRA or 6-FAM—of the dually labeled A•B−5 was linear with increasing DNA quantity. These data show that the TAMRA and 6-FAM fluorescence of these constructs can be used to quantify the relative proportions of the substrate and cleavage products. Representative gels for quantifying Ape1 cleavage are shown in -II.
Ape1 cleavage of abasic sites in partial and full duplex oligonucleotides
Ape1 activity was first determined using double-stranded A1•B, containing only a single abasic site. Fifty picograms Ape 1 cleaved ~80% of 2.5 pmol A1•B in 20 min under standard conditions. We then tested whether Ape1 action on a cluster-containing 51mer assembled from component oligomers requires ligation of those components. The cluster-containing substrate we used as a comparison standard was authentic A1•B−5, obtained as 51mer from the supplier: Ape1 cleaved 79.3 ± 4% of the substrate, comparable to the normal A1•B duplex. Notably, the enzyme had similar activities on the complete, assembled duplex (A1•BaBbBc; ), whether it was ligated (76.6 ± 5% cleaved) or not (72.7 ± 1%), suggesting that the neighboring SSBs in this context had no significant effect on Ape1 incision capacity.
We also evaluated Ape1 activity on the A-strand abasic site of three sub-components: Ape1 had no activity on single-stranded A1, as found previously by Wilson et al
), although we note that secondary structure can influence incision activity of Ape1 on single-stranded AP-containing oligonucleotides (29
). It cleaved ~40% of A1•Bb (a partial duplex with a 21-bp central duplex and two 15-bp single-stranded ends) and a similar level of A1•BaBb (partial duplex with a 3′ overhang of 15 bp). These results show that Ape1 can cleave a substantial range of cluster configurations. They further show that even gapped regions outside the immediate cluster region are not severe impediments to Ape1 cleavage at a central abasic site.
Cluster complexity and Ape1 cleavage
To test whether the number of lesions in a cluster determined its susceptibility to Ape1 action, we first measured Ape1 cleavage of a two-lesion cluster shown previously to be readily cleaved on both strands by this enzyme (7
). -I shows a diagram of three negative polarity clusters [B-strand abasic site(s) located 5′ to the base opposite the central A-strand abasic site]: the two-lesion A1•B−5 construct, and two three-lesion clusters formed by the addition of a second abasic site, producing A1•B−5−7 and A1•B−5−9 (). The A1•B−5−7 construct contains an additional abasic site on the B strand 2 nt from the −5 site, whereas the additional abasic site in the A1•B−5−9 duplex is 4 nt from the −5 site. The graph in -I shows the quantitative data for cleavage of these constructs by 25 pg Ape1. The rates of cleavage of the A1 abasic site in the simpler two-lesion cluster and more complex three-lesion cluster are quite similar, with the abasic site in the two-lesion cluster actually being cleaved slightly more slowly than in the three-lesion cluster.
In contrast, the positive polarity abasic clusters [B-strand abasic site(s) located 3′ to base opposite the central abasic site on strand A] are all resistant to Ape1 cleavage (Panel II, ). The insert shows the data for cleavage of three such cluster configurations by 250 pg Ape1 (the two-lesion A1•B+1 and the three-lesion clusters A1•B+1+4 and A1•B+1+6; ). The two-lesion cluster and the three-lesion cluster A1•B+1+4 were both cleaved slowly, but the A1•B+1+6 construct was resistant to cleavage.
Since these data indicated a strong polarity effect on susceptibility of abasic sites in clusters to cleavage, we investigated the cleavage of bipolar clusters, i.e. those whose B strand contains one abasic site situated in the positive direction and one in the negative direction from the abasic site on A1 (see the diagram in -III). The graph in -III shows that treatment with high concentrations of Ape1 (i.e. 250 pg) was required to produce cleavage of the very closely spaced three-lesion cluster A1•B+1−1 and of the A1•B+4−1 cluster. No cleavage of the A1•B+1−5 cluster could be detected.
Cleavage of A or B strands in specific clustered damages
Panel I of shows that the A1-abasic site in A1•B−5 is readily cleaved by Ape1, indicating that Ape1 action would produce a SSB. However, considerable previous data showed that many two-lesion bistranded clusters could be converted to DSBs (9
). To determine whether Ape1 action on the A1•B−5 cluster produces a DSB, we assessed the cleavage of the abasic sites on strand B as well as strand A. -I shows that in the two-lesion cluster A1•B−5, the abasic sites on the A and the B strands are cleaved readily. -II shows that the addition to the cluster in A1•B−5 of another lesion can have a striking effect on specific strand cleavage, depending on the configuration of the multiple lesions. In the A1•B−5−9 cluster, the A strand and both B strand abasic sites are cleaved (-III). However, in the more closely spaced cluster A1•B−5−7, both abasic sites on the B strand are cleaved only slowly (-II), yet the A-strand abasic site is cleaved rapidly. We also examined Ape1 cleavage of the same B-strand lesion configurations, but with no lesions on the A strand, i.e. a unistranded multiply damaged site or tandem cluster. The comparable unistranded tandem clusters on strand B to the bistranded A1•B−5−9 were cleaved about twice as well as the bistranded cluster. Further, the comparable tandem cluster to A1•B−5−7 was cleaved about three times as well as the bistranded A1•B-5-7.
Examination of A-strand abasic site cleavage in positive polarity clusters (-II; and for a representative gel -II) showed the resistance of the abasic site to Ape1 action. The presence of the B-strand abasic site appears to inhibit A-strand cleavage. However, this did not preclude the possibility that the abasic sites in the B strand were susceptible to Ape1 cleavage, and thus a SSB could be generated. Panel IV of shows that at high Ape1 levels, the B-strand abasic site of A1•B+1 is cleaved, generating the 5′ and 3′ ends of B as shown in the insert. Thus this cluster configuration leads to the slow production of a SSB (or gap). Likewise, although the A-strand abasic sites in A1•B+1+4 (-V) and in A1•B+1+6 (-VI) are poorly cleaved, high levels of Ape1 cleaved both B-strand abasic sites in both constructs, thus generating SSBs. Ape1 cleavage of comparable unistranded tandem clusters, as seen in strand B of the two substrates above, was 4–10 times higher than that of the bistranded positive polarity clusters (data not shown).
Likewise, -I shows that in bipolar clusters, high Ape1 levels produce moderate A-strand abasic site cleavage that is accompanied by substantial cleavage of both B-strand abasic sites. In A1•B+4−1, cleavage of B-strand abasic sites clusters are substantial at high Ape1 levels (-II), whereas in A1•B+1−5, production of the 3′-labeled end is substantially slower than of the 5′ labeled end product (-III). In A1•B+1−5, the virtual absence of A-strand cleavage precludes the generation of DSBs. In A1•B+4−1, a low level of DSBs could be generated slowly (-III). Similarly to the positive and negative polarity clusters, the comparable tandem unistranded clusters were cleaved 3–8 times better than the corresponding bistranded bipolar cluster.
High levels of SSBs in both A and B strands probably reflect the generation of DSBs. However, in constructs with only partial cleavage of one or both strands, the outcome cannot be determined by examination of individual strand cleavage using denaturing gels. To test whether SSBs or DSBs were generated by Ape1 action, we treated the cluster-containing oligonucleotide duplexes with Ape1 and dispersed the resulting products on nondenaturing polyacrylamide gels for detection of double-strand cleavage products. -I shows that the negative polarity clusters (A1•B−5, A1•B−5−7 and A1•B−5−9) are all cleaved by Ape1 to produce DSBs. The three-abasic site complex clusters were converted to DSBs with approximately the same kinetics as the two-lesion abasic clusters.
In contrast, two of the positive abasic site clusters were quite resistant to conversion to DSBs (-II). Even in reactions employing 250 pg Ape1 (shown in the insert), both the two-lesion cluster A1•B+1 and the three-lesion cluster A1•B+1+4 were resistant to DSB induction. DSBs were slowly induced at the three-lesion cluster A1•B+1+6.
Low levels of Ape1 produce little cleavage of bipolar clusters. The outcome of treatment of a bipolar cluster with high levels of Ape1 depends strongly on the configuration of the cluster (-III). None were rapidly cleaved to DSBs at Ape1 levels comparable to those readily generating DSBs in the negative polarity clusters. However, at high Ape1 levels (250 pg), generation of DSBs in both the A1•B+1-1 and A1•B+4−1 constructs was detectable. The A1•B+1−5 cluster was resistant to DSB cleavage.