To investigate the effect of nucleosomes on nucleotide excision repair in humans, we prepared a mononucleosome containing a (6-4) photoproduct in the nucleosome core and examined its repair with the reconstituted human excision nuclease system and with cell extracts. Nucleosomal DNA is repaired at a rate of about 10% of that for naked DNA in both systems. These results are in agreement with in vivo data showing a considerably slower rate of repair of overall genomic DNA relative to that for transcriptionally active DNA. Furthermore, our results indicate that the first-order packing of DNA in nucleosomes is a primary determinant of slow repair of DNA in chromatin.Nucleotide excision repair is a general repair system for removing virtually all types of lesions from DNA and is the sole repair mechanism for eliminating bulky base adducts (54). The repair reaction is initiated by dual incisions bracketing the lesion which release damage in the form of 24-to 32-nucleotide-long oligomers in humans (20) and in Saccharomyces cerevisiae (16). In a biochemically defined human system, 15 polypeptides in six repair factors, XPA, RPA, XPC, TFIIH, XPG, and XPF-ERCC1, are necessary and sufficient to excise the damage from naked DNA (43, 44). However, the physiological substrate for nucleotide excision is chromatin, and hence it is conceivable that in addition to the six general repair factors other enzymes which make lesions in chromatin accessible to the excision nuclease proper play an important role in genomic DNA repair.In vivo studies with both yeast and mammalian cells have revealed that the organization of DNA within chromatin has a strong negative effect on its repairability by the nucleotide excision repair system (37, 68). Similarly, in vivo studies have shown that transcribed DNA is repaired preferentially (4), and since transcription is invariably associated with significant chromatin remodeling (70, 81), it has been inferred that the various activators, coactivators, and remodeling and accessibility factors which play essential roles in transcription may play equally prominent roles in excision repair (37). The availability of a defined human excision nuclease system has made it possible to investigate the effect of chromatin structure on DNA repair. To do this, we used a mononucleosome as the substrate for human excision nuclease. We find that the nucleosome severely inhibits damage recognition and excision by both the purified human excision nuclease and mammalian cell extracts (CEs). MATERIALS AND METHODSDNA substrate. The structure of the 136-bp DNA substrate containing a unique T(6-4)T photoproduct is schematically shown in Fig. 1. The substrate DNA was prepared as described previously (45, 61). For footprinting experiments and to detect 5Ј incision, the DNA was terminally radiolabeled with 32 P at the 5Ј end of the damage-containing strand. To detect excision (dual incision) and for electrophoretic mobility shift experiments, the substrate was internally radiolabeled with 32 P at the fourth phosphodiester bon...

To investigate the role of chromatin remodeling in nucleotide excision repair, we prepared mononucleosomes with a 200-bp duplex containing an acetylaminofluorene-guanine (AAF-G) adduct at a single site. DNase I footprinting revealed a well-phased nucleosome structure with the AAF-G adduct near the center of twofold symmetry of the nucleosome core. This mononucleosome substrate was used to examine the effect of the SWI/SNF remodeling complex on the activity of human excision nuclease reconstituted from six purified excision repair factors. We found that the three repair factors implicated in damage recognition, RPA, XPA, and XPC, stimulate the remodeling activity of SWI/SNF, which in turn stimulates the removal of the AAF-G adduct from the nucleosome core by the excision nuclease. This is the first demonstration of the stimulation of nucleotide excision repair of a lesion in the nucleosome core by a chromatin-remodeling factor and contrasts with the ACF remodeling factor, which stimulates the removal of lesions from internucleosomal linker regions but not from the nucleosome core.Nucleotide excision repair (excision repair) is a multistep and all-purpose repair system which removes all DNA lesions, including UV photoproducts and alkylated and oxidized bases, from DNA (32, 52). The basic steps of this repair system include damage recognition, dual incision, excision (12), repair synthesis, and ligation. The excision of damage in human cells by dual incision is carried out by six repair factors, RPA, XPA, XPC, TFIIH, XPG, and XPF-ERCC1 (4,29,30). Similarly, the Saccharomyces cerevisiae homologs of these six factors are necessary and sufficient for dual incision (8). The basic enzymology of excision repair in both mammalian and yeast cells has been determined in considerable detail with naked DNA substrates (4,8,29,30,48). However, the natural substrate of this repair system in vivo is chromatin, and there have been only limited studies on the molecular mechanisms of excision repair of DNA damage in the nucleosome or chromatin in vitro (9,17,23,45).The nucleosome is the fundamental repeating unit of chromatin and constitutes the first order of DNA compaction in the nucleus. A nucleosome consists of two structurally different parts, the core particle and the linker. The nucleosome core particle consists of about 145 bp of DNA wrapped around the histone octamer. The nucleosome core particles are joined by the linker, consisting of approximately 50 bp of DNA associated with a linker histone to form the "beads-on-a-string" structure (16, 50). Packaging of DNA into the nucleosome has strong negative effects on essentially all DNA transactions, including replication, recombination, repair, and transcription (16, 41, 44); these effects have been best characterized with respect to transcriptional regulation. The development of an in vitro excision repair assay (12) and the reconstitution of the excision reaction in a defined six-factor system (29) have provided the opportunity to investigate the effect of DNA compaction in ...

SignificanceProtozoal proteasome is a validated target for antimalarial drug development, but species selectivity of reported inhibitors is suboptimal. Here we identify inhibitors with improved selectivity for malaria proteasome β5 subunit over each active subunit of human proteasomes. These compounds kill the parasite in each stage of its life cycle. They interact synergistically with a β2 inhibitor and with artemisinin. Resistance to the β5 inhibitor arose through a point mutation in the nonproteolytic β6 subunit. The same mutation made the mutant strain more sensitive to a β2 inhibitor and less fit to withstand irradiation. These findings reveal complex interplay among proteasome subunits and introduce the prospect that combined inhibition of β2 and β5 subunits can afford synergy and thwart resistance.

CDC-like kinase phosphorylation of serine/arginine-rich proteins is central to RNA splicing reactions. Yet, the genomic network of CDC-like kinase-dependent RNA processing events remains poorly defined. Here, we explore the connectivity of genomic CDC-like kinase splicing functions by applying graduated, short-exposure, pharmacological CDC-like kinase inhibition using a novel small molecule (T3) with very high potency, selectivity, and cell-based stability. Using RNA-Seq, we define CDC-like kinase-responsive alternative splicing events, the large majority of which monotonically increase or decrease with increasing CDC-like kinase inhibition. We show that distinct RNA-binding motifs are associated with T3 response in skipped exons. Unexpectedly, we observe dose-dependent conjoined gene transcription, which is associated with motif enrichment in the last and second exons of upstream and downstream partners, respectively. siRNA knockdown of CLK2-associated genes significantly increases conjoined gene formation. Collectively, our results reveal an unexpected role for CDC-like kinase in conjoined gene formation, via regulation of 3′-end processing and associated splicing factors.