UV-Induced DNA Damage and Mutagenesis in Chromatin

Abstract

UV radiation induces photolesions that distort the DNA double helix and, if not repaired, can cause severe biological consequences, including mutagenesis or cell death. In eukaryotes, both the formation and repair of UV damage occur in the context of chromatin, in which genomic DNA is packaged with histones into nucleosomes and higher order chromatin structures. Here, we review how chromatin impacts the formation of UV photoproducts in eukaryotic cells. We describe the initial discovery that nucleosomes and other DNA binding proteins induce characteristic "photofootprints" during the formation of UV photoproducts. We also describe recent progress in genomewide methods for mapping UV damage, which echoes early biochemical studies, and highlights the role of nucleosomes and transcription factors in UV damage formation and repair at unprecedented resolution. Finally, we discuss our current understanding of how the distribution and repair of UV-induced DNA damage influence mutagenesis in human skin cancers.

Figure 1. UV photofootprint of nucleosome core DNA

(a) Distributions of UV-induced (6-4)PDs and CPDs in nucleosomal DNA. Nucleosome core DNA was digested with T4 DNA polymerase-exonuclease before and after DNA photolyase treatment and the digestion profiles are shown. Lanes [CPD&(6-4)PD], no photoreversal; lanes [(6-4)PD Only], photoreversal with DNA photolyase. N, UV-irradiated nucleosome; D, UV-irradiated naked DNA. [See reference (21) for details.] (b) Laser densitometer scans of T4 DNA polymerase-exonuclease digestion profiles of UV-irradiated nucleosomes (NCP), and UV-irradiated naked DNA (DNA). NCP panel, values denote the scan absorbance (in arbitrary units) vs. distance (in bases) from the 5’ end of core DNA. Arrow represents position of the nucleosome dyad axis. DNA panel, same as NCP, except that core DNA was first isolated and then irradiated with UV light. [See reference (20) for details.]

Figure 2. Model of the nucleosome UV photofootprint

Formation of UV-induced CPD dimers occurs more frequently at ‘out’ rotational settings (indicated by bright blue lightning bolts) than at ‘in’ rotational settings (faint blue lightning bolts) in nucleosomal DNA. The relative level of local DNA mobility is represented by a color scale based on the B-factor from the 1id3 nucleosome structure, where DNA regions with a high B-factor (i.e., high mobility) are colored red, and DNA regions with a relatively low B-factor (i.e., low mobility) are colored blue. The image was generated using Pymol to visualize the nucleosome structure 1id3 (87). Only one gyre of the nucleosome DNA is depicted.

Figure 3. Nucleosome positioning power is affected by DNA sequence [data from reference (9)]

Nucleosomes reconstituted from two different nucleosome-positioning sequences (256 bp sea urchin 5S rDNA and 264 bp Widom- 601.3b sequence) are shown on a native gel. Multiple bands are shown for nucleosomes reconstituted with the relatively low affinity 5S DNA sequence. The multiplicity of bands reflects nucleosome positioning at many different positions in this sequence [schematically represented on left by 5S rDNA (thick vertical arrow) on 256 bp fragment (thin vertical line) and several NCP positions, from strongest binding translational position (shaded oval) to weaker binding positions (blue and red dashed lines)]. By contrast, nucleosome reconstitution with the high affinity 601.3b sequence shows only one dominant position. Lane M, 100 bp DNA size standard. Lanes D, naked DNA. Lanes N, reconstituted nucleosomes. [Adapted from Figure 3 of reference (9).]

Figure 4. The dynamic changes of nucleosomal DNA revealed by FRET

(a) Schematic diagram of DNA transient unwrapping dynamics in the nucleosome. The green internal disk represents histone octamer. Protein binding site is indicated as dark spot (black) on DNA (purple). [Adapted from: (88)]. (b) Locations of donor (Cy3) and acceptor (Cy5) dyes on the 147 bp 601 DNA sequence used for FRET. Upper panel shows locations of dyes on the naked DNA. The distance between the Cy3 and Cy5 on the naked DNA is ~27 nm, well beyond R₀ (~5 nm) for the Cy3-Cy5 pair. Lower panel shows the dye locations on 601 NCPs. The distance between the dyes is ~3 nm in NCPs, significantly less than R₀, yielding efficient energy transfer from donor to acceptor. The NCP model was generated from the crystal structure in PDB (accession number 1KX5). (c) Emission spectra revealed that energy transfer was only seen in the NCPs (blue) but not in naked DNA (pink). (d) FRET analysis demonstrates enhanced nucleosome unwrapping dynamics by UV damage. The 601 DNA was irradiated with different UV doses before reconstituted into NCPs. NCP Samples were excited at 515 nm and emission spectra recorded from 550 nm to 700 nm. Less efficient energy transfer is shown by increased donor emission and decreased acceptor emission, with increased UV doses. [Panels b-d are taken from reference (39).]

Figure 5. A method for measuring UV damage at the nucleotide level

(a) Schematic of the high-resolution UV damage mapping technique. Cells are UV irradiated and DNA is isolated immediately after UV treatment or at different repair times. The target DNA fragment is released from genomic or plasmid DNA by restriction enzymes. CPDs are incised by T4 endo V and converted to single strand breaks. A biotinylated DNA probe containing the complementary sequence to the 3’ end of one strand is annealed to the target DNA fragment. The annealed DNA is purified with streptavidin magnetic beads, and the target DNA is end-labeled with Sequenase and [³²P]-dATP, using the poly(dT) tract on the probe as the extension template. The radio-labeled products are separated on a sequencing gel. (b) Representative gel showing CPD sites and their repair in the GAL1-10 gene sequence under repressed (Glu) and activated (Gal) conditions. Yeast cells were irradiated with 50 J/m² UV light and allowed to repair for different times. Lanes labeled as ‘U’ indicate samples without UV irradiation. The left two lanes are A+G and C+T sequence markers generated by Maxam–Gilbert sequencing procedure. [Adapted from reference (45).]

Figure 6. Mapping UV damage in the yeast genome using the CPD-seq method

(A) Schematic of CPD-seq [adapted from (67)]. After fragmentation of genomic DNA isolated from UV-irradiated yeast cells, the trP1 adaptor (brown), containing a 3′ dideoxy group (denoted ‘dd’), is ligated to the free DNA ends. The remaining free 3′-OHs are further blocked by terminal transferase using ddATP as the substrate. DNA is subsequently digested with T4 endo V and APE1, to generate new 3′-OHs (green) specifically at CPD sites. The A adaptor (orange) is ligated to the free 3′-OH (green) immediately upstream of the CPD site. The resulting CPD library is briefly amplified with primers complementary to the trP1 and A adaptors and subjected to next-generation sequencing. Sequencing reads are mapped to the reference yeast genome to identify genomic locations and dinucleotide sequences of the CPDs. (B) CPD formation in vivo is enhanced at outward rotational settings (positions denoted by vertical dashed lines), but repressed at inward rotational settings in strongly positioned nucleosomes across the yeast genome. The scaled ratio of CPD formation in UV irradiated yeast (in vivo) relative to UV-irradiated naked DNA is plotted for the plus and minus strands of ~10,000 strongly positioned yeast nucleosomes. Green triangles indicate the locations of previously identified peaks of CPD formation (from the 3′ side of the CPD lesion) in mammalian chromatin, based on reference (20).

Figure 7. Effects of transcription factor binding on UV lesion formation, repair, and cancer mutations

The genome-wide analysis of UV lesion repair in relation to transcription factor binding sites (TFBS) show potentially complex correlations with the position of melanoma mutations. A general reduction of NER as measured by XR-seq in normal human fibroblasts (NHF1 cells) (black line) occurs in TFBS. This correlates with an increased mutation density in these areas in sequenced human melanomas (orange line). However, larger regions flanking the TFBS contain both low NER activity and low melanoma mutation density, suggesting a complex relationship occurs among these parameters, which may in part be explained by differences in lesions formation. XR-seq and melanoma mutation data were obtained from reference (86).