A combinatorial system to examine the enzymatic repair of multiply damaged DNA substrates

Abstract

DNA damage drives genetic mutations that underlie the development of cancer in humans. Multiple pathways have been described in mammalian cells which can repair this damage. However, most work to date has focused upon single lesions in DNA. We present here a combinatorial system which allows assembly of duplexes containing single or multiple types of damage by ligating together six oligonucleotides containing damaged or modified bases. The combinatorial system has dual fluorescent labels allowing examination of both strands simultaneously, in order to study interactions or competition between different DNA repair pathways. Using this system, we demonstrate how repair of oxidative damage in one DNA strand can convert a mispaired T:G deamination intermediate into a T:A mutation. We also demonstrate that slow repair of a T:G mispair, relative to a U:G mispair, by the human methyl-binding domain 4 DNA glycosylase provides a competitive advantage to competing repair pathways, and could explain why CpG dinucleotides are hotspots for C to T mutations in human tumors. Data is also presented that suggests repair of closely spaced lesions in opposing strands can be repaired by a combination of short and long-patch base excision repair and simultaneous repair of multiply damage sites can potentially lead to lethal double strand breaks.

Figure 1.

Assembly of a combinatorial system to examine the repair of multiply damaged DNA. (A) Oligo sequences used in the ligation reaction and representative gel showing unligated FAM labeled strand (lane 1), unligated Cy5 labeled strand (lane 2), post ligation reaction products (lane 3), and post gel purification product (lane 4). A superscript ‘P’ denotes a 5′ phosphate. (B) Full length oligo sequence with restriction enzyme cut sites. Sites containing modified or replaced nucleotides are labeled ‘X’, ‘Y’, and ‘Z.’ This convention is used to denote each unique multiply damaged substrate ‘X:Y_Z’ (e.g. C:G_G) in the supplement. BsrI has two potential cut sites denoted BsrI-1 and BsrI-2. A cut at BsrI-1 produces a 65-base green band while a cut at BsrI-2 produces a 37-base green band and a 15-base red band. HaeIII produces 39 base green and red bands. HpaII produces 33- and 43-base green and red bands, respectively.

Figure 2.

Characterization of a substrate containing complex damage. Glycosylases (hyTDG and hOGG1) or restriction endonucleases (BsrI, HpaII and HaeIII) were tested against a multiply damaged substrate. The T in the upper strand was mispaired with G. An 8-oxoguanine ('8') in the lower strand was paired with C. Cleavage by hyTDG and Tth produced a 33-base band, hOGG1 and APE1 a 38-base band, BsrI 65-base and 15-base bands. While HpaII cannot cleave a mispair containing duplex, HaeIII was able to cleave the upper strand producing a 39-base band but not the lower strand containing 8-oxoguanine.

Figure 3.

Examination of the repair of the upper and lower strands by the short-patch (SP) BER pathway. Potential restriction endonuclease cleavage sites for repair products are diagramed at the top for the upper strand (A) or lower strand (B). Nucleotidesinserted by pol β are underlined. SP-BER of the upper strand (A) was initiated by hyTDG, and for the lower strand (B) by hOGG1. Cleavage by hyTDG and APE1 produced a 33-base band (A, lane 4) while hOGG1 and APE1 produced a 38-base band (B, lane 4). Restriction endonuclease cleavage of the post-repair products confirmed that the T:G (A) was converted to C:G (HpaII: 43-base and 33-base bands A, lane 8) and the 8-oxoguanine (‘8’):C was repaired to C:G (HaeIII: 39-base bands B, lane 8).

Figure 4.

Examination of DNA repair by strand displacement synthesis and the long-patch (LP) BER pathway. The multiply damaged substrate and potential restriction endonuclease cut sites are shown with the top middle oligonucleotide. Repair was initiated in the upper strand by hyTDG (A, lane 3) or in the lower strand by hOGG1 (B, lane 3). Following repair, substrates were probed with restriction endonucleases. Cleavage by HpaII (A, lane 9) confirmed that the T:G mispair was converted to C:G. Cleavage by HaeII (B, lane 9) confirmed that the 8oxoG:C was repaired to G:C.

Figure 5.

LP-BER repair of 8oxoG can result in demethylation of nearby CpG sites. In the configuration shown above, 8oxoG ('8') is on the 5′ side of a T:G mispair, which could arise from the deamination of 5mC. The BER pathway was initiated by the removal of 8oxoG ('8') by hOGG1 (lane 2). Repair synthesis by pol β resulted in the insertion of G (lane 5), the replacement of the mispaired T with C (lane 7), and completion of the repair of the upper strand (lane 8). The CCGG HpaII site was initially blocked by the presence of the T:G mispair. However, after repair synthesis, the T:G was converted to C:G which allowed HpaII cleavage (lane 9). This result showed that BER can also function as an active demethylation pathway.

Figure 6.

Competition between short-patch (SP-BER) and long-patch (LP-BER) repair can occur on the lower strand. dNTPs denotes an equimolar amount of dCTP, dGTP, dATP and dTTP. Repair of the 8-oxoguanine ('8') in the lower strand by SP- or LP-BER pathways with restoration of the full-length sequence was demonstrated in lanes 2 to 4. Post-repair products were examined in lanes 5-7 with hyTDG. The SP-BER repair of the 8oxoG in the lower strand did not alter the T:G mispair, which was confirmed by hyTDG cleavage (lane 5). However, LP-BER of the 8oxoG in the lower strand converted the T:G mispair into a T:A base pair, which was not cleaved by hyTDG (lane 6). Competition between SP and LP-BER (lane 7) revealed predominant cleavage with hyTDG, indicating repair occurred mostly by SP-BER. However, hyTDG cleavage cannot reveal low levels of any LP-BER product. Post-repair products were also probed with BsrI (lanes 8-10). Cleavage of the SP-product with BsrI generated a 65-base green band, whereas the LP-product generated a 37-base band. BsrI cleavage of the products of the competition reaction revealed two bands, both SP- and LP-BER, with SP-BER occuring 2.5 times more than LP-BER.

Figure 7.

Competition between long-patch repair (LP-BER) of upper and lower strands can lead to a double strand break. (A) Diagram of LP-BER initiated on both strands resulting in double strand breaks with underlined nucleotides inserted by pol β. (B) Repair of both strands was initiated with hOGG1 and UDG. (C) Repair with hOGG1 and the relatively slow enzyme hTDG. Cleavage by UDG or hTDG and APE1 produced a 33-base band while hOGG1 and APE1 produced a 38-base band.

Figure 8.

Slow repair of a T:G mispair alters repair outcome of LP-BER competition. Competition between U and 8oxoG repair is shown in (A) whereas competition between T and 8oxoG is shown in (B). When repair was initiated with hMBD4 and hOGG1 (A, lane 3), the U-containing strand was partially cleaved, whereas the 8oxoG-containing strand was completely cleaved. Subsequent repair with pol β and all dNTPs resulted in predominant repair of both strands with some double strand breaks (A, lane 6). The excision of T:G by hMBD4 (B, lane 2) was much slower than U (A, lane 2). Subsequent repair with pol β and dNTPs generated full length sequences (B, lane 6). BsrI cleavage (A, lane 7) indicated conversion of the U:G to U:A or T:A, whereas in B, lane 7, the predominant repair product was T:A. Panel B revealed that most of the 8oxoG-containing strand was repaired before hMBD4 could initiate repair of the T:G mispair.

Figure 9.

Competition between SP-BER and LP-BER simultaneously in both upper and lower strands is compared with different combinations of repair enzymes. Competition between UDG and hOGG1 is shown in panels A and C, and between hTDG and hOGG1 in panels B and D. In panel A and B, SP-BER was completed by E. coli ligase, whereas in panels C and D, T4 ligase was used. In panel A, both strands were completely cleavage by UDG and hOGG1. Addition of pol β and dGTP allowed complete repair of the lower strand (A, lane 5). Interestingly, simultaneous addition ofdGTP and dCTPresulted in less repair of the lower strand and significant amounts of truncated strands (A, lane 8). Replacement of UDG with hTDG allowed preferential repair of the lower strand. Similar results were obtained when E. coli ligase was replaced with T4 ligase (C&D).

Figure 10.

Competition for repair of a multiply damaged substrate using all human enzymes results in a double-strand break. In the system shown above, repair was initiated first on the upper strand by incubation with hUNG2 (lane 2). Addition of pol β, dCTP and hLIGIII/XRCC1 resulted in 60% completion of repair by the SP-BER pathway (lane 3). Repair was then initiated on the lower strand by incubation with hOGG1 (lane 4) which removed the 8oxoG ('8'). Addition of pol β, dGTP and hLIGIII/XRCC1 allowed the completion of repair (57%) by SP-BER. When repair was initiated on both upper and lower strands simultaneously, both strands were cleaved. Addition of pol β, dNTPs and hLIGIII/XRCC1 resulted in partial elongation of both strands, with no ligation. This is in contrast to the data presented with E. coli ligase and T4 ligase shown in Figure 9.