Fritz Thoma

Light and dark in chromatin repair: repair of UV-induced DNA lesions by photolyase and nucleotide excision repair.

Nucleotide excision repair (NER) and DNA repair by photolyase in the presence of light (photoreactivation) are the major pathways to remove UV‐induced DNA lesions from the genome, thereby preventing mutagenesis and cell death. Photoreactivation was found in many prokaryotic and eukaryotic organisms, but not in mammals, while NER seems to be universally distributed. Since packaging of eukaryotic DNA in nucleosomes and higher order chromatin structures affects DNA structure and accessibility, damage formation and repair are coupled intimately to structural and dynamic properties of chromatin. Here, I review recent progress in the study of repair of chromatin and transcribed genes. Photoreactivation and NER are discussed as examples of how an individual enzyme and a complex repair pathway, respectively, access DNA lesions in chromatin and how these two repair processes fulfil complementary roles in removal of UV lesions. These repair pathways provide insight into the structural and dynamic properties of chromatin and suggest how other DNA repair processes could work in chromatin.

Nucleosome structure and positioning modulate nucleotide excision repair in the non-transcribed strand of an active gene.

Nucleotide excision repair (NER) is a major pathway to remove pyrimidine dimers (PDs), a class of DNA lesions generated by ultraviolet light. Since folding of DNA into nucleosomes restricts its accessibility and since transcription and DNA repair require access to DNA, nucleosome structure and positioning as well as the transcriptional state may affect DNA repair. We recently determined the chromatin structure of the yeast URA3 gene at high resolution and found multiple positions of nucleosomes as well as strand‐ and site‐specific variation in DNA accessibility to DNase I (internal protected regions). Here, the same high‐resolution primer extension technique was used to investigate NER of PDs in the URA3 gene of a minichromosome in vivo. In the non‐transcribed strand (NTS), fast repair correlates with PD locations in linker DNA and towards the 5′ end of a positioned nucleosome. Slow repair correlates with the internal protected region of the nucleosome. This repair heterogeneity reflects a modulation of NER by positioned nucleosomes in the NTS. NER in the transcribed strand (TS) is fast, less heterogeneous and shows no correlation with chromatin structure. Apparently, transcription‐coupled repair overrides chromatin modulation of NER in the TS. Heterogeneity in NER generated by chromatin structure on the NTS may contribute to heterogeneity in mutagenesis.

Rapid accessibility of nucleosomal DNA in yeast on a second time scale

Packaging DNA in nucleosomes and higher‐order chromatin structures restricts its accessibility and constitutes a barrier for all DNA transactions including gene regulation and DNA repair. How and how fast proteins find access to DNA buried in chromatin of living cells is poorly understood. To address this question in a real time in vivo approach, we investigated DNA repair by photolyase in yeast. We show that overexpressed photolyase, a light‐dependent DNA‐repair enzyme, recognizes and repairs UV‐damaged DNA within seconds. Rapid repair was observed in various nucleosomal regions of the genome including inactive and active genes and repressed promoters. About 50% of cyclobutane pyrimidine dimers were removed in 5 s, >80% in 90 s. Heterochromatin was repaired within minutes, centromeres were not repaired. Consistent with fast conformational transitions of nucleosomes observed in vitro, this rapid repair strongly suggests that spontaneous unwrapping of nucleosomes rather than histone dissociation or chromatin remodeling provides DNA access. The data impact our view on the repressive and dynamic nature of chromatin and illustrate how proteins like photolyase can access DNA in structurally and functionally diverse chromatin regions.

SITE-SPECIFIC REPAIR OF CYCLOBUTANE PYRIMIDINE DIMERS IN A POSITIONED NUCLEOSOME BY PHOTOLYASE AND T4 ENDONUCLEASE V IN VITRO

Since genomic DNA is folded into nucleosomes, and DNA damage is generated all over the genome, a central question is how DNA repair enzymes access DNA lesions and how they cope with nucleosomes. To investigate this topic, we used a reconstituted nucleosome (HISAT nucleosome) as a substrate to generate DNA lesions by UV light (cyclobutane pyrimidine dimers, CPDs), and DNA photolyase and T4 endonuclease V (T4‐endoV) as repair enzymes. The HISAT nucleosome is positioned precisely and contains a long polypyrimidine region which allows one to monitor formation and repair of CPDs over three helical turns. Repair by photolyase and T4‐endoV was inefficient in nucleosomes compared with repair in naked DNA. However, both enzymes showed a pronounced site‐specific modulation of repair on the nucleosome surface. Removal of the histone tails did not substantially enhance repair efficiency nor alter the site specificity of repair. Although photolyase and T4‐endoV are different enzymes with different mechanisms, they exhibited a similar site specificity in nucleosomes. This implies that the nucleosome structure has a decisive role in DNA repair by exerting a strong constraint on damage accessibility. These findings may serve as a model for damage recognition and repair by more complex repair mechanisms in chromatin.

TATA‐binding protein promotes the selective formation of UV‐induced (6‐4)‐photoproducts and modulates DNA repair in the TATA box

DNA‐damage formation and repair are coupled to the structure and accessibility of DNA in chromatin. DNA damage may compromise protein binding, thereby affecting function. We have studied the effect of TATA‐binding protein (TBP) on damage formation by ultraviolet light and on DNA repair by photolyase and nucleotide excision repair in yeast and in vitro. In vivo, selective and enhanced formation of (6‐4)‐photoproducts (6‐4PPs) was found within the TATA boxes of the active SNR6 and GAL10 genes, engaged in transcription initiation by RNA polymerase III and RNA polymerase II, respectively. Cyclobutane pyrimidine dimers (CPDs) were generated at the edge and outside of the TATA boxes, and in the inactive promoters. The same selective and enhanced 6‐4PP formation was observed in a TBP–TATA complex in vitro at sites where crystal structures revealed bent DNA. We conclude that similar DNA distortions occur in vivo when TBP is part of the initiation complexes. Repair analysis by photolyase revealed inhibition of CPD repair at the edge of the TATA box in the active SNR6 promoter in vitro, but not in the GAL10 TATA box or in the inactive SNR6 promoter. Nucleotide excision repair was not inhibited, but preferentially repaired the 6‐4PPs. We conclude that TBP can remain bound to damaged promoters and that nucleotide excision repair is the predominant pathway to remove UV damage in active TATA boxes.

Kinetochores prevent repair of UV damage in Saccharomyces cerevisiae centromeres

ABSTRACT Centromeres form specialized chromatin structures termed kinetochores which are required for accurate segregation of chromosomes. DNA lesions might disrupt protein-DNA interactions, thereby compromising segregation and genome stability. We show that yeast centromeres are heavily resistant to removal of UV-induced DNA lesions by two different repair systems, photolyase and nucleotide excision repair. Repair resistance persists in G1- and G2/M-arrested cells. Efficient repair was obtained only by disruption of the kinetochore structure in a ndc10-1 mutant, but not in cse4-1 and cbf1Δ mutants. Moreover, UV photofootprinting and DNA repair footprinting showed that centromere proteins cover about 120 bp of the centromere elements CDEII and CDEIII, including 20 bp of flanking CDEIII. Thus, DNA lesions do not appear to disrupt protein-DNA interactions in the centromere. Maintaining a stable kinetochore structure seems to be more important for the cell than immediate removal of DNA lesions. It is conceivable that centromeres are repaired by postreplication repair pathways.

Induction of a G1-S checkpoint in fission yeast

Entry into S phase is carefully regulated and, in most organisms, under the control of a G1-S checkpoint. We have previously described a G1-S checkpoint in fission yeast that delays formation of the prereplicative complex at chromosomal replication origins after exposure to UV light (UVC). This checkpoint absolutely depends on the Gcn2 kinase. Here, we explore the signal for activation of the Gcn2-dependent G1-S checkpoint in fission yeast. If some form of DNA damage can activate the checkpoint, deficient DNA repair should affect the length of the checkpoint-induced delay. We find that the cell-cycle delay differs in repair-deficient mutants from that in wild-type cells. However, the duration of the delay depends not only on the repair capacity of the cells, but also on the nature of the repair deficiency. First, the delay is abolished in cells that are deficient in the early steps of repair. Second, the delay is prolonged in repair mutants that fail to complete repair after the incision stage. We conclude that the G1-S delay depends on damage to the DNA and that the activating signal derives not from the initial DNA damage, but from a repair intermediate(s). Surprisingly, we find that activation of Gcn2 does not depend on the processing of DNA damage and that activated Gcn2 alone is not sufficient to delay entry into S phase in UVC-irradiated cells. Thus, the G1-S delay depends on at least two different inputs.

Nucleosome positioning, nucleotide excision repair and photoreactivation in Saccharomyces cerevisiae.

The position of nucleosomes on DNA participates in gene regulation and DNA replication. Nucleosomes can be repressors by limiting access of factors to regulatory sequences, or activators by facilitating binding of factors to exposed DNA sequences on the surface of the core histones. The formation of UV induced DNA lesions, like cyclobutane pyrimidine dimers (CPDs), is modulated by DNA bending around the core histones. Since CPDs are removed by nucleotide excision repair (NER) and photolyase repair, it is of paramount importance to understand how DNA damage and repair are tempered by the position of nucleosomes. In vitro, nucleosomes inhibit NER and photolyase repair. In vivo, nucleosomes slow down NER and considerably obstruct photoreactivation of CPDs. However, over-expression of photolyase allows repair of nucleosomal DNA in a second time scale. It is proposed that the intrinsic abilities of nucleosomes to move and transiently unwrap could facilitate damage recognition and repair in nucleosomal DNA.

Contributions of Histone H3 Nucleosome Core Surface Mutations to Chromatin Structures, Silencing and DNA Repair

Histone H3 mutations in residues that cluster in a discrete region on the nucleosome surface around lysine 79 of H3 affect H3-K79 methylation, impair transcriptional silencing in subtelomeric chromatin, and reveal distinct contributions of histone H3 to various DNA-damage response and repair pathways. These residues might act by recruitment of silencing and DNA-damage response factors. Alternatively, their location on the nucleosome surface suggests a possible involvement in nucleosome positioning, stability and nucleosome interactions. Here, we show that the yeast H3 mutants hht2-T80A, hht2-K79E, hht2-L70S, and hht2-E73D show normal nucleosome positioning and stability in minichromosomes. However, loss of silencing in a subtelomeric URA3 gene correlates with a shift of the promoter nucleosome, while nucleosome positions and stability in the coding region are maintained. Moreover, the H3 mutants show normal repair of UV lesions by photolyase and nucleotide excision repair in minichromosomes and slightly enhanced repair in the subtelomeric region. Thus, these results support a role of those residues in the recruitment of silencing proteins and argue against a general role in nucleosome organization.