Accommodation and Repair of a UV Photoproduct in DNA at Different Rotational Settings on the Nucleosome Surface

Željko M Svedružić,Michael J Smerdon,Chenbo Wang,Joseph V Kosmoski

doi:10.1074/jbc.m509478200

Željko M Svedružić, Michael J Smerdon + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m509478200

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Abstract

Cyclobutane-thymine dimers (CTDs), the most common DNA lesion induced by UV radiation, cause 30 degrees bending and 9 degrees unwinding of the DNA helix. We prepared site-specific CTDs within a short sequence bracketed by strong nucleosome-positioning sequences. The rotational setting of CTDs over one turn of the helix near the dyad center on the histone surface was analyzed by hydroxyl radical footprinting. Surprisingly, the position of CTDs over one turn of the helix does not affect the rotational setting of DNA on the nucleosome surface. Gel-shift analysis indicates that one CTD destabilizes histone-DNA interactions by 0.6 or 1.1 kJ/mol when facing away or toward the histone surface, respectively. Thus, 0.5 kJ/mol energy penalty for a buried CTD is not enough to change the rotational setting of sequences with strong rotational preference. The effect of rotational setting on CTD removal by nucleotide excision repair (NER) was examined using Xenopus oocyte nuclear extracts. The NER rates are only 2-3 times lower in nucleosomes and change by only 1.5-fold when CTDs face away or toward the histone surface. Therefore, in Xenopus nuclear extracts, the rotational orientation of CTDs on nucleosomes has surprisingly little effect on rates of repair. These results indicate that nucleosome dynamics and/or chromatin remodeling may facilitate NER in gaining access to DNA damage in nucleosomes.

Highlights

The effect of chromatin structure on repair of UV damage was first implicated by the findings of Wilkins and Hart [7]
The Glucocorticoid hormone Response Element (GRE) sequence containing a Cyclobutane-thymine dimers (CTDs) lesion was bracketed by multiple repeats of the TG motif [33] in an attempt to modulate the rotational setting of CTDs near the nucleosome dyad axis [31]
Within strong nucleosome positioning sequences like the TG motif, CTDs are accommodated by the intrinsic DNA rotational setting on the nucleosome surface that is similar to undamaged DNA

Summary

MATERIALS AND METHODS

Synthetic DNA Substrates—Cis-syn cyclobutyl-thymidine dimers were prepared as phosphoramidites by organic synthesis, as previously described [24], and incorporated into oligonucleotides at the desired sequence by solid phase synthesis. The interaction between DNA-binding proteins and the DNA phosphate backbone [34] We have used this reaction to compare the rotational setting (Fig. 1) of undamaged DNA on the histone surface in nucleosomes with that of DNA molecules containing CTDs at different sites 5 nucleotides apart (i.e. one half turn of the DNA helix, Fig. 1). From the ratio of the initial slopes of the curves, we calculate that DNA with CTD-OUT is repaired at about half the rate when bound to nucleosomes (and facing away from the histones) relative to free DNA (Fig. 5B and TABLE TWO) This result is similar to earlier results from our laboratory for CTDs facing out near the dyad center of nucleosomes [18, 37]. Values represent the mean Ϯ one S.D. for at least three independent experiments

68 Ϯ 6 67 Ϯ 5

Findings

DISCUSSION