Abstract
Human chromatin-associated protein extracts were examined for endonucleolytic activity on a defined 132-base pair DNA substrate containing a single, site-specific 4,5'-8-trimethylpsoralen plus long wavelength ultraviolet light-induced furan side or pyrone side monoadduct or interstrand cross-link. These extracts produced incisions on both the 3' and 5' sides of each of these lesions. The distance between the 3' and 5' incisions at sites of a furan side monoadduct or cross-link was 9 nucleotides, and at sites of a pyrone side monoadduct or cross-link it was 17 nucleotides. Incisions on the 3' side of both types of furan side and pyrone side adducts were similar and were either at the fourth or fifth phosphodiester bond from the adducted thymine, depending upon the adduct. However, greater differences were observed between sites of 5' incision. This incision occurred at the fifth and sixth phosphodiester bonds from the adducted thymine at sites of furan side monoadducts and cross-links, respectively, and at the 13th and 14th phosphodiester bonds at sites of pyrone side monoadducts and cross-links, respectively. Thus, direct analysis of sites of endonucleolytic incision reveals that the location of sites of incision on TMP-adducted substrates depends upon the type of adduct present.
Highlights
Repair of DNA interstrand cross-links is critical for a number of cellular processes such as transcription and DNA replication and is important for the maintenance of genetic integrity and cellular survival
Psoralen-DNA adducts are formed in three stages; psoralen first intercalates into the DNA duplex in a noncovalent manner and upon photoreaction with UVA light, forms either a furan side or a pyrone side monoadduct with the 5,6-double bond of a pyrimidine, which the majority of the time is a thymine
Each complex contains proteins involved in damage recognition, chromatin interaction, and endonucleolytic incision, and each has been demonstrated to be involved in the repair process by its ability to correct the repair defect in repair-deficient cells when introduced into them via electroporation [28, 29]
Summary
Repair of DNA interstrand cross-links is critical for a number of cellular processes such as transcription and DNA replication and is important for the maintenance of genetic integrity and cellular survival. We have isolated from the nuclei of normal human cells a chromatin-associated DNA endonuclease complex, pI 4.6, which recognizes and incises DNA containing TMP or 8-methoxypsoralen plus UVA interstrand cross-links and another, pI 7.6, which recognizes and incises DNA containing psoralen monoadducts [22, 23, 25, 27]. We describe the construction of a 132base pair oligonucleotide substrate that contains a centrally placed single, site-specific TMP monoadduct or interstrand cross-link These substrates are so constructed that endonucleolytic activity on either the furan side or the pyrone side of each type of adduct may be selectively studied. Our results show that these extracts make incisions in DNA on
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