Cockayne syndrome B protein: connection between repair, transcription and chromatin structure

DNA is the carrier of the genetic instructions in all living organisms. Its integrity is of vital importance for a faithful transmission of the genetic information and for the proper fimctioning of cellular processes. However, the DNA molecule is susceptible to alterations caused by both intrinsic chemical instability (e.g. deatninatioll, depurinatioll etc.) and by a wide variety of environmental and endogenous compounds. The most prominent DNAwdamaging physical agents arc ultraviolet (UV) light and ionizing radiation (X-rays and y-rays). DNA damage can disturb cellular processes and can have severe consequences on human health. Its direct effect at the cellular level is inhibition of vital processes, most notably transcription, replication and cell cycle progression. Accumulation of lesions in DNA can either lead to cell death by apoptosis or to permanent mutation

Biochemical and biological characterization of wild-type and ATPase-deficient Cockayne syndrome B repair protein

Cockayne syndrome (CS) is a nucleotide excision repair disorder characterized by sun (UV) sensitivity and severe developmental problems. Two genes have been shown to be involved: CSA and CSB. Both proteins play an essential role in preferential repair of transcription-blocking lesions from active genes. In this study we report the purification and characterization of baculovirus-produced HA-His6-tagged CSB protein (dtCSB), using a highly efficient three-step purification protocol. Microinjection of dtCSB protein in CS-B fibroblasts shows that it is biologically functional in vivo. dtCSB exhibits DNA-dependent ATPase activity, stimulated by naked as well as nucleosomal DNA. Using structurally defined DNA oligonucleotides, we show that double-stranded DNA and double-stranded DNA with partial single-stranded character but not true single-stranded DNA act as efficient cofactors for CSB ATPase activity. Using a variety of substrates, no overt DNA unwinding by dtCSB could be detected, as found with other SNF2/SWI2 family proteins. By site-directed mutagenesis the invariant lysine residue in the NTP-binding motif of CSB was substituted with a physicochemically related arginine. As expected, this mutation abolished ATPase activity. Surprisingly, the mutant protein was nevertheless able to partially rescue the defect in recovery of RNA synthesis after UV upon microinjection in CS-B fibroblasts. These results indicate that integrity of the conserved nucleotide-binding domain is important for the in vivo function of CSB but that also other properties independent from ATP hydrolysis may contribute to CSB biological functions

The Cockayne syndrome B protein, involved in transcription-coupled DNA repair, resides in an RNA polymerase II-containing complex

Transcription-coupled repair (TCR), a subpathway of nucleotide excision repair (NER) defective in Cockayne syndrome A and B (CSA and CSB), is responsible for the preferential removal of DNA lesions from the transcribed strand of active genes, permitting rapid resumption of blocked transcription. Here we demonstrate by microinjection of antibodies against CSB and CSA gene products into living primary fibroblasts, that both proteins are required for TCR and for recovery of RNA synthesis after UV damage in vivo but not for basal transcription itself. Furthermore, immunodepletion showed that CSB is not required for in vitro NER or transcription. Its central role in TCR suggests that CSB interacts with other repair and transcription proteins. Gel filtration of repair- and transcription-competent whole cell extracts provided evidence that CSB and CSA are part of large complexes of different sizes. Unexpectedly, there was no detectable association of CSB with several candidate NER and transcription proteins. However, a minor but significant portion (10-15%) of RNA polymerase II was found to be tightly associated with CSB. We conclude that within cell-free extracts, CSB is not stably associated with the majority of core NER or transcription components, but is part of a distinct complex involving RNA polymerase II. These findings suggest that CSB is implicated in, but not essential for, transcription, and support the idea that Cockayne syndrome is due to a combined repair and transcription deficiency

Plant–environment interactions through a functional traits perspective: a review of Italian studies

Italy is among the European countries with the greatest plant diversity due to both a great environmental heterogeneity and a long history of man–environment interactions. Trait-based approaches to ecological studies have developed greatly over recent decades worldwide, although several issues concerning the relationships between plant functional traits and the environment still lack sufficient empirical evaluation. To draw insights on the association between plant functional traits and direct and indirect human and natural pressures on the environmental drivers, this article summarizes the existing knowledge on this topic by reviewing the results of studies performed in Italy adopting a functional trait approach on vascular plants, bryophytes and lichens. Although we recorded trait measurements for 1418 taxa, our review highlighted some major gaps in plant traits knowledge: Mediterranean ecosystems are poorly represented; traits related to belowground organs are still overlooked; traits measurements for bryophytes and lichens are lacking. Finally, intraspecific variation has been little studied at community level so far. We conclude by highlighting the need for approaches evaluating trait–environment relationship at large spatial and temporal scales and the need of a more effective contribution to online databases to tie more firmly Italian researchers to international scientific networks on plant traits