Effect of damage type on stimulation of human excision nuclease by SWI/SNF chromatin remodeling factor - PubMed (original) (raw)
Effect of damage type on stimulation of human excision nuclease by SWI/SNF chromatin remodeling factor
Ryujiro Hara et al. Mol Cell Biol. 2003 Jun.
Abstract
To investigate the repair of different types of DNA lesions in chromatin, we prepared mononucleosomes containing an acetylaminofluorene-guanine adduct (AAF-G), a (6-4) photoproduct, or a cyclobutane pyrimidine dimer (CPD) and measured the repair of these lesions by reconstituted 6-factor human excision nuclease. We find that incorporation into nucleosomes inhibits the repair of CPD more severely than repair of the AAF-G adduct and the (6-4) photoproduct. Equally important, we find that SWI/SNF stimulates the removal of AAF-G and (6-4) photoproduct but not of CPD from nucleosomal DNA. These results shed new light on the low rate of repair of CPDs in human cells in vivo.
Figures
FIG. 1.
Substrates. (A) Schematic of substrate construction from six oligomers by ligation. The arm fragments AF1 to AF4 are 95, 94, 90, and 91 nt in length, respectively. The length and sequences of damaged oligomers and complementary sequence (CF) for UV- and AAF-damaged substrates are shown. Asterisks indicate the positions of the radiolabel in duplexes used for footprinting and for the excision assay. (B) Sequence of the T<>T or (6-4) photoproduct-containing duplex. The triangle indicates position of the photoproduct.
FIG. 2.
Analysis of damage-containing mononucleosomes by polyacrylamide gel electrophoresis. Naked DNA (D) or nucleosomes (N) carrying the indicated lesions were separated on a 5% nondenaturing polyacrylamide gel and were visualized by autoradiography. CPD, cyclobutane pyrimidine dimer.
FIG. 3.
DNase I footprinting of naked DNA and nucleosomal (Nuc) substrates. (A) Footprints of the AAF-G substrate and the corresponding undamaged DNA (UM). (B) Footprints of the two UV photoproducts and of unmodified DNA of identical sequence. The nucleosome reconstitution mixtures were treated with DNase I and were directly loaded on a 5% nondenaturing polyacrylamide gel. The free and nucleosomal DNA bands were located by autoradiography, were recovered from the gel, and were then separated on 6% denaturing polyacrylamide gels that were autoradiographed. The ∼10-nt periodicity in nucleosomal DNA is indicated by open arrows, and the positions of G-AAF and the UV photoproducts are marked.
FIG. 4.
Inhibition of excision of three types of DNA lesions by nucleosomes. The indicated substrates, either as naked DNA or in nucleosomes (Nuc), were incubated with 6-factor reconstituted excision nuclease for 3 h at 30°C, and the products were separated on an 8% denaturing polyacrylamide gel. The figure shows an autoradiogram of the gel. The levels of excision were quantified by PhosphorImager and the ImageQuant system and are indicated at the bottoms of all lanes.
FIG. 5.
Effect of SWI/SNF on repair of three different lesions in mononucleosomes. Nucleosomal DNAs (30 pM) were incubated with reconstituted excision nuclease in the absence or presence of SWI/SNF (0.48 nM) for the indicated times, and the reaction products were separated on 8% polyacrylamide gels. The top panels show representative autoradiograms of excision gels. In the bottom panel the level of excision as percentage of input substrates is plotted from three independent experiments; the bars indicate standard errors.
References
- Aalfs, J. D., and R. E. Kingston. 2000. What does ‘chromatin remodeling’ mean? Trends Biochem. Sci. 25:548-555. - PubMed
- Bessho, T., A. Sancar, L. H. Thompson, and M. P. Thelen. 1997. Reconstitution of human excision nuclease with recombinant XPF·ERCC1 complex. J. Biol. Chem. 272:3833-3837. - PubMed
- Branum, M. E., J. T. Reardon, and A. Sancar. 2001. DNA repair excision nuclease attacks undamaged DNA: a potential source of spontaneous mutations. J. Biol. Chem. 276:25421-25426. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources