Nucleotide excision repair of the 5 S ribosomal RNA gene assembled into a nucleosome

A-175-base pair fragment containing the Xenopus borealis somatic 5 S ribosomal RNA gene was used as a model system to determine the effect of nucleosome assembly on nucleotide excision repair (NER) of the major UV photoproduct (cyclobutane pyrimidine dimer (CPD)) in DNA. Xenopus oocyte nuclear extracts were used to carry out repair in vitro on reconstituted, positioned 5 S rDNA nucleosomes. Nucleosome structure strongly inhibits NER at many CPD sites in the 5 S rDNA fragment while having little effect at a few sites. The time course of CPD removal at 35 different sites indicates that >85% of the CPDs in the naked DNA fragment have t(12) values <2 h, whereas <26% of the t(12) values in nucleosomes are <2 h, and 15% are >8 h. Moreover, removal of histone tails from these mononucleosomes has little effect on the repair rates. Finally, nucleosome inhibition of repair shows no correlation with the rotational setting of a 14-nucleotide-long pyrimidine tract located 30 base pairs from the nucleosome dyad. These results suggest that inhibition of NER by mononucleosomes is not significantly influenced by the rotational orientation of CPDs on the histone surface, and histone tails play little (or no) role in this inhibition.     

Nucleotide excision repair in chromatin: damage removal at the drop of a HAT

In an earlier review of our understanding of the mechanism of nucleotide excision repair (NER) we examined the process with respect to how it occurs in chromatin [1]. We described how much of our mechanistic understanding of NER was derived from biochemical studies that analysed the repair reaction in DNA substrates not representative of that which exists in the living cell. We pointed out that our efforts to understand how NER operates in chromatin had been hampered in part because of the well-known inhibition of NER that occurs when DNA is assembled into nucleosomes and used as the substrate to examine the repair reaction in vitro. Despite this technical bottleneck, we summarized the biochemical, genetic and cell-based studies which have provided insights into the molecular mechanism of NER in the cellular context. More recently, we revisited the topic of how UV induced DNA damage is repaired in chromatin. In this review we examined the commonly held view that depicts a struggle in which the DNA repair machinery battles to overcome the inhibitory effect of chromatin during the repair process. We suggested that in this interpretation of events, the DNA repair mechanisms might be described as 'tilting at windmills': fighting an imaginary foe [2]. We surmised that this scenario was overly simplistic, and we described an emerging picture in which the DNA repair process and chromatin remodeling were mechanistically linked and were in fact functioning cooperatively to organize the efficient removal of DNA damage from the genome. Here we discuss the latest findings, which contribute to the idea that DNA damage induced changes to chromatin represent an important way in which the DNA repair process is initiated and organized throughout the genome to promote the efficient removal of damage in response to UV radiation.     

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.     

NuA4 acetyltransferase is required for efficient nucleotide excision repair in yeast

The nucleotide excision repair (NER) pathway is critical for removing damage induced by ultraviolet (UV) light and other helix-distorting lesions from cellular DNA. While efficient NER is critical to avoid cell death and mutagenesis, NER activity is inhibited in chromatin due to the association of lesion-containing DNA with histone proteins. Histone acetylation has emerged as an important mechanism for facilitating NER in chromatin, particularly acetylation catalyzed by the Spt-Ada-Gcn5 acetyltransferase (SAGA); however, it is not known if other histone acetyltransferases (HATs) promote NER activity in chromatin. Here, we report that the essential Nucleosome Acetyltransferase of histone H4 (NuA4) complex is required for efficient NER in Saccharomyces cerevisiae. Deletion of the non-essential Yng2 subunit of the NuA4 complex causes a general defect in repair of UV-induced cyclobutane pyrimidine dimers (CPDs) in yeast; in contrast, deletion of the Sas3 catalytic subunit of the NuA3 complex does not affect repair. Rapid depletion of the essential NuA4 catalytic subunit Esa1 using the anchor-away method also causes a defect in NER, particularly at the heterochromatic HML locus. We show that disrupting the Sds3 subunit of the Rpd3L histone deacetylase (HDAC) complex rescued the repair defect associated with loss of Esa1 activity, suggesting that NuA4-catalyzed acetylation is important for efficient NER in heterochromatin.