Since IR induces clustered damage sites containing two or more lesions formed within one or two helical turns of the DNA double helix by a single radiation track, and the majority of studies to date have focussed on clusters containing two lesions, synthetic oligonucleotides have been designed containing a variety of three-lesions within a clustered damage site. We have extended understanding of the hierarchy of repair of clustered damage based on the finding that the reparability of an AP site(s) in three-lesion clusters depends upon the composition of the lesions within the cluster and whether its incision leads to potentially cytotoxic DSBs or are highly mutagenic in E. coli. For instance, the presence of 8-oxoG when present in a cluster with bistranded AP sites does not prevent the formation of DSB whereas with clusters containing two 8-oxoG in tandem opposite to an AP site, DSBs are not formed. Although a proportion of the mutations for clusters containing tandem 8-oxoG opposite to an AP site contain GC to AT transversions at both 8-oxoG sites, surprisingly the mutations occur predominantly at the 8-oxoG located closer to the 3′ terminus of the inserted oligonucleotide.
We have confirmed from the dramatic loss of colonies in wild-type E. coli
that DSBs form with clusters containing two AP sites, one on each strand. DSB formation was also confirmed when the clusters containing two bistranded AP sites were incubated with the nuclear extract, even in the case of those clusters which showed a ‘two population’ effect where some repair was also seen. These latter clusters showed a reduced efficiency for APE1 cleavage of the second AP site. With bistranded uracil clusters we see a rescue of colony survival in the ung1 E. coli
strain, since the lack of ung1 prevents DSB formation, implying that in wild-type bacteria the uracils are converted into AP sites, resulting ultimately in a DSB. This finding is also consistent with the formation of DSBs in wild-type E. coli
when the uracils had been converted to AP sites prior to transformation, and the findings with bistranded AP site clusters after transformation into yeast (29
) and bacterial (44
) strains deficient in various AP endonucleases. Evidence that the DSB arises through BER processing of the AP sites and not due to replication-induced DSB comes from the observation that when a single-strand nick (no loss of base) is present in close proximity to an 8-oxoG on the other strand, the nick is repaired before excision of 8-oxoG, minimising DSB formation (58
). In contrast, when the 8-oxoG is replaced by an AP site, a DSB rapidly forms as there is insufficient time for the nick to be repaired before rapid incision of the AP site. From the few surviving colonies in wild-type E. coli
with these clusters, the most prevalent mutations seen are small deletions at the site of one of the uracil residues. No mutations were seen at the site of 8-oxoG with these particular clusters.
In contrast, DSBs are not formed in E. coli
with clusters containing two tandem 8-oxoG lesions opposite a uracil/AP site. In bacteria there are two main glycosylases involved in the prevention of mutation arising from 8-oxoG; fpg that excises 8-oxoG when present within the DNA double helix, and mutY, that removes adenine residues misincorporated opposite 8-oxoG lesions, with a smaller role for mutT which hydrolyses 8-oxoGTP to 8-oxoGMP in the nucleotide pool to prevent its integration into the DNA sequence. The mutation frequency for single 8-oxoG is low in wild-type and MutY E. coli,
whereas a dramatic increase was seen following transformation of clustered damage containing tandem 8-oxoG lesions opposite a uracil/AP site into mutY
bacteria, consistent with mutY being the main glycosylase to protect against mutations at 8-oxoG (28
). The overall mutation frequency in mutY E. coli
is similar to that determined with the bistranded AP/8-oxoG containing clusters (40
). Surprisingly, the vast majority of the mutations with the clusters containing tandem 8-oxoG are GC:AT transversions, occurring at the 8-oxoG closer to the 3′-end of the inserted oligonucleotide. This preferential site for mutations reinforces the concept of a hierarchy of lesion excision within clustered damage sites previously shown biochemically (49
) with two tandem 8-oxoG lesions using cell extracts and purified proteins. The increased incidence of mutation at the more 3′ lesion implies that the other 8-oxoG, in our case the 8-oxoG at −2 within U/8-oxoG+2−2 and at +1 in U/8-oxoG+1+5, is excised preferentially. When two 8-oxoG residues are placed in tandem to each other and treated with purified Fpg (0.65 pmol) it was verified that the lesion located closer to the 5′ terminus of the oligonucleotide is preferentially incised (data not shown). Therefore, in our system we propose the following mechanisms of repair; the uracil residue is rapidly excised and the resulting AP site rapidly incised to leave a cluster containing a SSB and two tandem 8-oxoG lesions. The resultant SSB is slowly repaired as shown in cell extracts so that excision of 8-oxoG is retarded (23
). Once the SSB is rejoined one of the two 8-oxoG residues may then be excised, with the more 5′ lesion being excised preferentially. The resultant SSB from excision of one of the 8-oxoG residues would be repaired with reduced efficiency, prior to potential excision of the remaining 8-oxoG, which if still present at replication results in mutations. If the SSB resulting from excision of uracil is still present at replication, this would lead to loss off the SSB-containing strand (40
) and mutations at both 8-oxoG sites, as is indeed seen (). Clusters containing a SSB and two tandem 8-oxoG lesions did not show induction of DSBs as no loss of colonies was observed (A) so it is confirmed that excision of 8-oxoG occurs once the SSB on the opposing strand has been rejoined.
In support of this mechanism, it was confirmed using nuclear extracts that the rejoining of the SSB, arising from incision of the AP site in clusters containing two tandem 8-oxoG lesions opposing a single AP site (AP/8-oxoG+1+5, AP/8-oxoG+2−
5), is indeed retarded. We had previously shown that the AP site is incised with an efficiency that is 7.5x that of excision of 8-oxoG (31
) so under the conditions used 8-oxoG would not be expected to be excised to any great extent. Further, the resulting strand break from incision of the AP site would additionally retard excision of 8-oxoG by glycosylases. Using purified proteins, the ligation step involving ligase III was shown to be retarded and not polymerase β which efficiently adds a base into the break site of these clusters and removes the dRP residue on the 5′ terminus of the SSB, leaving a 5′-phosphate ready for ligation. With the cluster AP/8-oxoG+1+5, the subsequent ligation was not observed after incubation with nuclear extract. This observation was unexpected since with bistranded AP site/8-oxoG clusters (23
), ligation products were seen even when the AP site is at +1 to the 8-oxoG, suggesting that the presence of the second 8-oxoG lesion interferes greatly with the efficiency of ligase III. The footprint of ligase III is four base pairs 5′ and 14 base pairs 3′ to the SSB, on the break-containing strand (59
) and ligase I fully encircles the DNA double-helix (60
). The greater retarded ligation seen may reflect enzyme–DNA contacts on the opposing strand containing the tandem 8-oxoG. Further, ligase I may compensate for a loss of ligase III activity for all orientations with bistranded AP site/8-oxoG clusters, except when in the +1 position to each other (26
). The zinc finger and DNA binding domain of ligase III combine to form a novel intrinsic DNA sensing module which differs to ligase I which does not contain a zinc finger motif (61
Several studies have shown that cleavage of two bistranded AP sites is retarded when in the +1 to +3 orientation to each other (25
). This may in part explain the ‘two population’ effect seen with the clusters AP/AP+2 8-oxoG−2 and AP/AP+1 8-oxoG+5, when the two AP sites are in this orientation. Additionally, AP endonuclease is a structure specific binding enzyme that recognises the kink introduced into the DNA by the formation of an AP site and has a footprint on the lesion-containing strand of six base pairs 5′ and five base pairs 3′ to the AP site (62
). It also makes contact with a base on the opposing strand to the AP site to ensure that the active site of the enzyme is correctly positioned for the lesion to be repaired. If an AP site at either position +1 or +2 interferes with the contact made on the opposing strand it may cause cleavage retardation. Due to the lack of retardation of incision of the AP site found on strand 1, the 8-oxoG in tandem with it has no effect. However, incision of the remaining AP site is retarded possibly reflecting some interference of the APE1 contact due to the 8-oxoG and the SSB on the opposite strand. As a consequence the SSB may persist through to replication leading to a replication-induced DSB or to incorporation of adenine as the preferred nucleotide across from the non-coding AP site during both replication and transcription (63
In summary, novel insights are presented into how more complex clustered damage sites formed as a result of exposure to IR, provide a serious challenge to the repair machinery of the cell. It is not only the prompt formation of DSBs that have implications on cell survival but also the conversion of non-DSB clusters to DSBs during processing and attempted repair. With the three-lesion clusters we have enhanced understanding on the hierarchy of processing of tandem lesions when opposed to an AP site/SSB. Due to the complexity of these clustered damage sites, BER is compromised and as a result the mutation frequency is increased. The inefficient repair of such clustered damage sites has great biological significance due to the ultimate risk of tumourogenesis.