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Efficient processing of TFO-directed psoralen DNA interstrand crosslinks by the UvrABC nuclease.

Christensen LA, Wang H, Van Houten B, Vasquez KM - Nucleic Acids Res. (2008)

Bottom Line: Because different chemistries may alter the processing of TFO-directed ICLs, we investigated the effect of another type of triplex formed by purine-rich TFOs on the processing of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) ICLs by the UvrABC nuclease.In contrast to previous reports, the UvrABC nuclease cleaved the TFO-directed psoralen ICL with a greater efficiency than that of the psoralen ICL alone.Furthermore, the TFO was dissociated from its duplex binding site by UvrA and UvrB.

View Article: PubMed Central - PubMed

Affiliation: Department of Carcinogenesis, Science Park-Research Division, University of Texas MD Anderson Cancer Center, Smithville, TX, USA.

ABSTRACT
Photoreactive psoralens can form interstrand crosslinks (ICLs) in double-stranded DNA. In eubacteria, the endonuclease UvrABC plays a key role in processing psoralen ICLs. Psoralen-modified triplex-forming oligonucleotides (TFOs) can be used to direct ICLs to specific genomic sites. Previous studies of pyrimidine-rich methoxypsoralen-modified TFOs indicated that the TFO inhibits cleavage by UvrABC. Because different chemistries may alter the processing of TFO-directed ICLs, we investigated the effect of another type of triplex formed by purine-rich TFOs on the processing of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) ICLs by the UvrABC nuclease. Using an HMT-modified TFO to direct ICLs to a specific site, we found that UvrABC made incisions on the purine-rich strand of the duplex approximately 3 bases from the 3'-side and approximately 9 bases from the 5'-side of the ICL, within the TFO-binding region. In contrast to previous reports, the UvrABC nuclease cleaved the TFO-directed psoralen ICL with a greater efficiency than that of the psoralen ICL alone. Furthermore, the TFO was dissociated from its duplex binding site by UvrA and UvrB. As mutagenesis by TFO-directed ICLs requires nucleotide excision repair, the efficient processing of these lesions supports the use of triplex technology to direct DNA damage for genome modification.

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UvrABC incision assay with the radiolabel on the pyrimidine-rich strand of the target duplex. (A) Samples were incubated with 20 nM UvrA, 100 nM UvrB and 50 nM UvrC as described in the Materials and methods section and subjected to denaturing PAGE on a 6% gel. Lanes 1 and 2 contain TFO-ICL −/+ UvrABC. The insert contains a magnified view of the incision product. The incision product migrating just above 20 bases corresponds to cutting on the pyrimidine-rich strand of the target duplex. (B) Expected incision sites for UvrABC on the TFO-ICL substrate. The expected incision product is shown in red, the TFO is depicted in blue and incision sites are marked by arrows. A green star indicates the position of the radiolabel. The pyrimidine-rich and purine-rich strands of the target duplex are labeled py and pu, respectively. (C) UvrABC incision assay with the ICL-only substrate 5′-end-labeled on the pyrimidine-rich strand of the target duplex. Lanes 1 and 2 contain the F2650 positive control, and lanes 3 and 4 contain ICL-only substrate. (D) Expected incision sites for ICL-only substrate. The figure is marked as in (B). (E) UvrABC incision assay with substrate 3′-end-labeled on both strands of the target duplex. Lanes 1 and 2 contain TFO-ICL −/+ UvrABC. (F). Expected incision sites for the UvrABC nuclease on the TFO-ICL substrate. The figure is marked as in (B).
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Figure 4: UvrABC incision assay with the radiolabel on the pyrimidine-rich strand of the target duplex. (A) Samples were incubated with 20 nM UvrA, 100 nM UvrB and 50 nM UvrC as described in the Materials and methods section and subjected to denaturing PAGE on a 6% gel. Lanes 1 and 2 contain TFO-ICL −/+ UvrABC. The insert contains a magnified view of the incision product. The incision product migrating just above 20 bases corresponds to cutting on the pyrimidine-rich strand of the target duplex. (B) Expected incision sites for UvrABC on the TFO-ICL substrate. The expected incision product is shown in red, the TFO is depicted in blue and incision sites are marked by arrows. A green star indicates the position of the radiolabel. The pyrimidine-rich and purine-rich strands of the target duplex are labeled py and pu, respectively. (C) UvrABC incision assay with the ICL-only substrate 5′-end-labeled on the pyrimidine-rich strand of the target duplex. Lanes 1 and 2 contain the F2650 positive control, and lanes 3 and 4 contain ICL-only substrate. (D) Expected incision sites for ICL-only substrate. The figure is marked as in (B). (E) UvrABC incision assay with substrate 3′-end-labeled on both strands of the target duplex. Lanes 1 and 2 contain TFO-ICL −/+ UvrABC. (F). Expected incision sites for the UvrABC nuclease on the TFO-ICL substrate. The figure is marked as in (B).

Mentions: To determine whether the UvrABC nuclease incises the pyrimidine-rich strand of the crosslinked target duplexes, the 5′-end of the pyrimidine-rich strand was labeled and subjected to the UvrABC incision assay. For both TFO-ICL and ICL-only substrates 5′-incision efficiency on the pyrimidine-rich strand was <3% (Figure 4). However, an incision product of ∼220 bases was observed by denaturing PAGE in the sample containing the TFO-ICL substrate and the UvrABC nuclease (Figure 4A, lane 2), likely due to purine-rich strand incision. Similarly, incubation of the ICL-only substrate with UvrABC produced two products migrating between 140 and 150 bases (Figure 4C, lane 4), again suggesting that these products were the result of cutting on the purine-rich strand of the target duplex only (Figure 4B and D). Labeling of the 3′-ends likewise revealed an incision product of ∼220 bp (Figure 4E, lane 2), but no detectable product of ∼88 bases as might be expected with cutting on the 3′ side of the ICL on the pyrimidine-rich strand (Figure 4F). These results point to inefficient incision by UvrABC on the pyrimidine-rich strand compared to the purine-rich strand of the target duplex on both the TFO-ICL and the ICL-only substrates.Figure 4.


Efficient processing of TFO-directed psoralen DNA interstrand crosslinks by the UvrABC nuclease.

Christensen LA, Wang H, Van Houten B, Vasquez KM - Nucleic Acids Res. (2008)

UvrABC incision assay with the radiolabel on the pyrimidine-rich strand of the target duplex. (A) Samples were incubated with 20 nM UvrA, 100 nM UvrB and 50 nM UvrC as described in the Materials and methods section and subjected to denaturing PAGE on a 6% gel. Lanes 1 and 2 contain TFO-ICL −/+ UvrABC. The insert contains a magnified view of the incision product. The incision product migrating just above 20 bases corresponds to cutting on the pyrimidine-rich strand of the target duplex. (B) Expected incision sites for UvrABC on the TFO-ICL substrate. The expected incision product is shown in red, the TFO is depicted in blue and incision sites are marked by arrows. A green star indicates the position of the radiolabel. The pyrimidine-rich and purine-rich strands of the target duplex are labeled py and pu, respectively. (C) UvrABC incision assay with the ICL-only substrate 5′-end-labeled on the pyrimidine-rich strand of the target duplex. Lanes 1 and 2 contain the F2650 positive control, and lanes 3 and 4 contain ICL-only substrate. (D) Expected incision sites for ICL-only substrate. The figure is marked as in (B). (E) UvrABC incision assay with substrate 3′-end-labeled on both strands of the target duplex. Lanes 1 and 2 contain TFO-ICL −/+ UvrABC. (F). Expected incision sites for the UvrABC nuclease on the TFO-ICL substrate. The figure is marked as in (B).
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Show All Figures
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Figure 4: UvrABC incision assay with the radiolabel on the pyrimidine-rich strand of the target duplex. (A) Samples were incubated with 20 nM UvrA, 100 nM UvrB and 50 nM UvrC as described in the Materials and methods section and subjected to denaturing PAGE on a 6% gel. Lanes 1 and 2 contain TFO-ICL −/+ UvrABC. The insert contains a magnified view of the incision product. The incision product migrating just above 20 bases corresponds to cutting on the pyrimidine-rich strand of the target duplex. (B) Expected incision sites for UvrABC on the TFO-ICL substrate. The expected incision product is shown in red, the TFO is depicted in blue and incision sites are marked by arrows. A green star indicates the position of the radiolabel. The pyrimidine-rich and purine-rich strands of the target duplex are labeled py and pu, respectively. (C) UvrABC incision assay with the ICL-only substrate 5′-end-labeled on the pyrimidine-rich strand of the target duplex. Lanes 1 and 2 contain the F2650 positive control, and lanes 3 and 4 contain ICL-only substrate. (D) Expected incision sites for ICL-only substrate. The figure is marked as in (B). (E) UvrABC incision assay with substrate 3′-end-labeled on both strands of the target duplex. Lanes 1 and 2 contain TFO-ICL −/+ UvrABC. (F). Expected incision sites for the UvrABC nuclease on the TFO-ICL substrate. The figure is marked as in (B).
Mentions: To determine whether the UvrABC nuclease incises the pyrimidine-rich strand of the crosslinked target duplexes, the 5′-end of the pyrimidine-rich strand was labeled and subjected to the UvrABC incision assay. For both TFO-ICL and ICL-only substrates 5′-incision efficiency on the pyrimidine-rich strand was <3% (Figure 4). However, an incision product of ∼220 bases was observed by denaturing PAGE in the sample containing the TFO-ICL substrate and the UvrABC nuclease (Figure 4A, lane 2), likely due to purine-rich strand incision. Similarly, incubation of the ICL-only substrate with UvrABC produced two products migrating between 140 and 150 bases (Figure 4C, lane 4), again suggesting that these products were the result of cutting on the purine-rich strand of the target duplex only (Figure 4B and D). Labeling of the 3′-ends likewise revealed an incision product of ∼220 bp (Figure 4E, lane 2), but no detectable product of ∼88 bases as might be expected with cutting on the 3′ side of the ICL on the pyrimidine-rich strand (Figure 4F). These results point to inefficient incision by UvrABC on the pyrimidine-rich strand compared to the purine-rich strand of the target duplex on both the TFO-ICL and the ICL-only substrates.Figure 4.

Bottom Line: Because different chemistries may alter the processing of TFO-directed ICLs, we investigated the effect of another type of triplex formed by purine-rich TFOs on the processing of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) ICLs by the UvrABC nuclease.In contrast to previous reports, the UvrABC nuclease cleaved the TFO-directed psoralen ICL with a greater efficiency than that of the psoralen ICL alone.Furthermore, the TFO was dissociated from its duplex binding site by UvrA and UvrB.

View Article: PubMed Central - PubMed

Affiliation: Department of Carcinogenesis, Science Park-Research Division, University of Texas MD Anderson Cancer Center, Smithville, TX, USA.

ABSTRACT
Photoreactive psoralens can form interstrand crosslinks (ICLs) in double-stranded DNA. In eubacteria, the endonuclease UvrABC plays a key role in processing psoralen ICLs. Psoralen-modified triplex-forming oligonucleotides (TFOs) can be used to direct ICLs to specific genomic sites. Previous studies of pyrimidine-rich methoxypsoralen-modified TFOs indicated that the TFO inhibits cleavage by UvrABC. Because different chemistries may alter the processing of TFO-directed ICLs, we investigated the effect of another type of triplex formed by purine-rich TFOs on the processing of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) ICLs by the UvrABC nuclease. Using an HMT-modified TFO to direct ICLs to a specific site, we found that UvrABC made incisions on the purine-rich strand of the duplex approximately 3 bases from the 3'-side and approximately 9 bases from the 5'-side of the ICL, within the TFO-binding region. In contrast to previous reports, the UvrABC nuclease cleaved the TFO-directed psoralen ICL with a greater efficiency than that of the psoralen ICL alone. Furthermore, the TFO was dissociated from its duplex binding site by UvrA and UvrB. As mutagenesis by TFO-directed ICLs requires nucleotide excision repair, the efficient processing of these lesions supports the use of triplex technology to direct DNA damage for genome modification.

Show MeSH
Related in: MedlinePlus