Structure, function and regulation of CSB: A multi-talented gymnast

doi:10.1016/j.mad.2013.02.004

Mechanisms of Ageing and Development

Volume 134, Issues 5–6, May–June 2013, Pages 202-211

https://doi.org/10.1016/j.mad.2013.02.004 Get rights and content

Abstract

The Cockayne syndrome complementation group B protein, CSB, plays pivotal roles in transcription regulation and DNA repair. CSB belongs to the SNF2/SWI2 ATP-dependent chromatin remodeling protein family, and studies from many laboratories have revealed that CSB has multiple activities and modes of regulation. To understand the underlying mechanisms of Cockayne syndrome, it is necessary to understand how the biochemical activities of CSB are used to carry out its biological functions. In this review, we summarize our current knowledge of the structure, function and regulation of CSB, and discuss how these properties can impact the biological functions of this chromatin remodeler.

Highlights

► Mechanisms of CSB–chromatin interaction. ► ATP-dependent CSB activities. ► ATP-independent CSB activities. ► CSB modification and oligomerization.

Section snippets

CSB: A SNF2/SWI2 ATPase family member

CSB belongs to the SNF2/SWI2 family of ATPases (Fig. 1) (Flaus et al., 2006, Hopfner et al., 2012). These enzymes contain a central ATPase domain, which consists of seven conserved helicase motifs, and it is the homology between the ATPase domains that defines this protein family. The regions flanking the central ATPase domain are divergent between family members, and these regions often contain domains that are important for protein localization or auto-regulation (Fig. 1). The SNF2/SWI2

Functional insights from CSB mutations

CSB can be roughly divided into three parts: a central ATPase domain flanked by N-terminal and C-terminal regions (Fig. 1, Fig. 3). Within the N-terminal region there is an acidic-rich region of unknown function (Brosh et al., 1999, Troelstra et al., 1992), and the C-terminal region harbors a ubiquitin-binding domain (UBD) (Anindya et al., 2010). There are also two predicted nuclear localization sequences that lie on either side of the ATPase domain.

Human genetic studies have cataloged a number

Post-translation modification of the CSB protein

Post-translational protein modification is a fundamental mechanism that is used to regulate protein function. One of the most common modifications is phosphorylation, which occurs on serine, threonine and tyrosine residues. Proteomic approaches have revealed that CSB is phosphorylated on multiple serine residues (Dephoure et al., 2008, Matsuoka et al., 2007, Nousiainen et al., 2006, Yu et al., 2007). Five of these residues lie in the N-terminal region of CSB (serines 158, 429, 430, 486 and 489)

CSB oligomerization

The oligomeric state of a chromatin remodeler can have a profound impact on the remodeling outcome. For instance, the SWI/SNF remodeler, a large, multiple-subunit remodeling complex, functions as a monomer. SWI/SNF regulates DNA access by opening or occluding sites for factor binding and has an essential role in transcription regulation. In contrast, the ACF remodeling complex functions as a dimer (Racki et al., 2009). ACF plays an important role in creating equally spaced nucleosomes for the

Alteration of chromatin structure

Biochemical studies have demonstrated that the ATP hydrolysis activity of CSB can be stimulated by a variety of DNA substrates, which include double-stranded DNA fragments, stem-looped DNA, DNA fragments with splayed arms, plasmid DNA and nucleosomal DNA. Although there appears to be a large degree of degeneracy in the types of substrates that stimulate CSB's ATPase activity, the common feature of all these substrates is duplex DNA, and double-stranded DNA appears to be essential, as single

ssDNA annealing activity

In vitro biochemical studies have revealed that CSB facilities the annealing of single-stranded DNA (ssDNA), at a rate that is 25-fold faster than spontaneous annealing, and promotes strand exchange (Muftuoglu et al., 2006). CSB has also been shown to promote the annealing of DNA/RNA hybrids and RNA/RNA duplexes as well (Berquist and Wilson, 2009). Interestingly, the ssDNA annealing and strand exchange reactions do not require ATP. In fact, ATP was found to inhibit the strand annealing activity

Regulation of CSB–chromatin association

ATP-dependent chromatin remodelers interact with DNA in a sequence-independent manner; however, they do not impact chromatin structure randomly but function in spatially regulated manners. How do ATP-dependent chromatin remodelers know where to go? ATP-dependent chromatin remodelers can be recruited to their sites of action by different mechanisms, which include interactions with specifically modified histones, with histone variants or with sequence-specific transcription factors. These

What does CSB do once recruited to sites of DNA lesion-stalled transcription?

Transcription-coupled DNA repair is a multistep process and CSB likely participates in different steps (Hanawalt and Spivak, 2008). The mutation frequency decline protein, Mfd, is a bacterial ATPase that is believed to be the prokaryotic counterpart of CSB. Mfd plays an essential role in displacing DNA lesion-stalled stalled RNA polymerase and initiating repair protein recruitment through direct protein–protein interaction (Selby and Sancar, 1995). In comparison, CSB has not yet been found to

Future perspectives

From the in vitro and in vivo studies described above, it is clear that CSB performs like a multi-talented gymnast, displaying strength, balance and agility in a variety of competitive cellular events. These events include, but are not limited to, nuclear and mitochondrial transcription regulation, transcription-coupled nucleotide excision repair, base excision repair, autophagy and apoptosis (see other articles in this issue for comprehensive reviews on these topics). Although much progress

Acknowledgements

Work from the authors’ laboratory is supported by the US National Institutes of Health (GM 084983 to H.-Y. Fan).

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In the context of replication-independent ICL repair, it was shown that the presence of CSB stimulates the nuclease activity of hSNM1A in vitro [83]. CSB is suggested to play a role in chromatin remodelling and is part of the SWI12/SNF2 family of DNA-dependent ATPases [101]. Interestingly, in the in vitro system used in this study, CSB stimulated the activity of hSNM1A on DNA substrates that it, itself is unable to bind, thus suggesting this stimulation arises due to protein-protein interactions [83].
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Cockayne Syndrome Type A Protein Protects Primary Human Keratinocytes from Senescence
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Defects in Cockayne syndrome type A (CSA), a gene involved in nucleotide excision repair, cause an autosomal recessive syndrome characterized by growth failure, progressive neurological dysfunction, premature aging, and skin photosensitivity and atrophy. Beyond its role in DNA repair, the CSA protein has additional functions in transcription and oxidative stress response, which are not yet fully elucidated. Here, we investigated the role of CSA protein in primary human keratinocyte senescence. Primary keratinocytes from three patients with CS-A displayed premature aging features, namely premature clonal conversion, high steady-state levels of reactive oxygen species and 8-OH-hydroxyguanine, and senescence-associated secretory phenotype. Stable transduction of CS-A keratinocytes with the wild-type CSA gene restored the normal cellular sensitivity to UV irradiation and normal 8-OH-hydroxyguanine levels. Gene correction was also characterized by proper restoration of keratinocyte clonogenic capacity and expression of clonal conversion key regulators (p16 and p63), decreased NF-κB activity and, in turn, the expression of its targets (NOX1 and MnSOD), and the secretion of senescence-associated secretory phenotype mediators. Overall, the CSA protein plays an important role in protecting cells from senescence by facilitating DNA damage processing, maintaining physiological redox status and keratinocyte clonogenic ability, and reducing the senescence-associated secretory phenotype-mediated inflammatory phenotype.
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CSB is well conserved from yeast to human and belongs to the Swi2/Snf2 family of nucleosome remodelers that all share the signature seven helicase motifs in their core ATPase domains [101,102,110,111]. CSB protein has DNA-dependent ATPase, DNA translocase, as well as chromatin remodeling activities [101,111–115]. CSB recruits CSA to the sites of arrested Pol II [109].
Eukaryotic transcription-coupled nucleotide excision repair (TC-NER) is a pathway that removes DNA lesions capable of blocking RNA polymerase II (Pol II) transcription from the template strand. This process is initiated by lesion-arrested Pol II and the recruitment of Cockayne Syndrome B protein (CSB). In this review, we will focus on the lesion recognition steps of eukaryotic TC-NER and summarize the recent research progress toward understanding the structural basis of Pol II-mediated lesion recognition and Pol II-CSB interactions. We will discuss the roles of CSB in both TC-NER initiation and transcription elongation. Finally, we propose an updated model of tripartite lesion recognition and verification for TC-NER in which CSB ensures Pol II-mediated recognition of DNA lesions for TC-NER.
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Cockayne syndrome (CS) is a premature aging disorder characterized by developmental defects, multisystem progressive degeneration and sensitivity to ultraviolet light. CS is divided into two primary complementation groups, A and B, with the CSA and CSB proteins presumably functioning in DNA repair and transcription. Using laser microirradiation and confocal microscopy, we characterized the nature and regulation of the CS protein response to oxidative DNA damage, double-strand breaks (DSBs), angelicin monoadducts and trioxsalen interstrand crosslinks (ICLs). Our data indicate that CSB recruitment is influenced by the type of DNA damage and is most rapid and robust as follows: ICLs > DSBs > monoadducts > oxidative lesions. Transcription inhibition reduced accumulation of CSB at sites of monoadducts and ICLs, but it did not affect recruitment to (although slightly affected retention at) oxidative damage. Inhibition of histone deacetylation altered the dynamics of CSB assembly, suggesting a role for chromatin status in the response to DNA damage, whereas the proteasome inhibitor MG132 had no effect. The C-terminus of CSB and, in particular, its ubiquitin-binding domain were critical to recruitment, while the N-terminus and a functional ATPase domain played a minor role at best in facilitating protein accumulation. Although the absence of CSA had no effect on CSB recruitment, CSA itself localized at sites of ICLs, DSBs and monoadducts but not at oxidative lesions. Our results reveal molecular components of the CS protein response and point to a major involvement of complex lesions in the pathology of CS.
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UVSSA, the product of the UVSS-A causative gene, forms a complex with USP7 (22, 23) and interacts with CSB and Pol II in a CSA- and UV light-dependent manner (22, 25). Posttranslational modifications play important roles in the functions of CSB (26). It has been reported that CSB is ubiquitinated and degraded in a UV light- and CSA-dependent manner (27).
Cockayne syndrome (CS) is a recessive disorder that results in deficiencies in transcription-coupled nucleotide excision repair (TC-NER), a subpathway of nucleotide excision repair, and cells from CS patients exhibit hypersensitivity to UV light. CS group B protein (CSB), which is the gene product of one of the genes responsible for CS, belongs to the SWI2/SNF2 DNA-dependent ATPase family and has an ATPase domain and an ubiquitin-binding domain (UBD) in the central region and the C-terminal region, respectively. The C-terminal region containing the UBD is essential for the functions of CSB. In this study, we generated several CSB deletion mutants and analyzed the functions of the C-terminal region of CSB in TC-NER. Not only the UBD but also the C-terminal 30-amino acid residues were required for UV light resistance and TC-NER. This region was needed for the interaction of CSB with RNA polymerase II, the translocation of CS group A protein to the nuclear matrix, and the association of CSB with chromatin after UV irradiation. CSB was modified by small ubiquitin-like modifier 2/3 in a UV light-dependent manner. This modification was abolished in a CSB mutant lacking the C-terminal 30 amino acid residues. However, the substitution of lysine residues in this region with arginine did not affect SUMOylation or TC-NER. By contrast, substitution of a lysine residue in the N-terminal region with arginine decreased SUMOylation and resulted in cells with defects in TC-NER. These results indicate that both the most C-terminal region and SUMOylation are important for the functions of CSB in TC-NER.

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Structure, function and regulation of CSB: A multi-talented gymnast

Abstract

Highlights

Section snippets

CSB: A SNF2/SWI2 ATPase family member

Functional insights from CSB mutations

Post-translation modification of the CSB protein

CSB oligomerization

Alteration of chromatin structure

ssDNA annealing activity

Regulation of CSB–chromatin association

What does CSB do once recruited to sites of DNA lesion-stalled transcription?

Future perspectives

Acknowledgements

Molecular Cell

Journal of Biological Chemistry

Journal of Molecular Biology

Molecular Cell

Cell

Journal of Biological Chemistry

Current Opinion in Structural Biology

Molecular Cell

Molecular Cell

Journal of Biological Chemistry

Mutation Research

Current Opinion in Structural Biology

Journal of Biological Chemistry

Molecular Cell

American Journal of Human Genetics

Journal of Biological Chemistry

Gene

Molecular Cell

Journal of Biological Chemistry

Journal of Biological Chemistry

Cell

Cell

Neuron

DNA Repair (Amst)

Molecular Cell

Molecular Cell

Strand pairing by Rad54 and Rad51 is enhanced by chromatin

Genes and Development

Mot1, a global repressor of RNA polymerase II transcription, inhibits TBP binding to DNA by an ATP-dependent mechanism

Genes and Development

Cockayne syndrome group B protein interacts with TRF2 and regulates telomere length and stability

Nucleic Acids Research

Human Cockayne syndrome B protein reciprocally communicates with mitochondrial proteins and promotes transcriptional elongation

Nucleic Acids Research

SMARCAL1 catalyzes fork regression and Holliday junction migration to maintain genome stability during DNA replication

Genes and Development

The ATPase domain but not the acidic region of Cockayne syndrome group B gene product is essential for DNA repair

Molecular Biology of the Cell

Functional consequences of mutations in the conserved SF2 motifs and post-translational phosphorylation of the CSB protein

Nucleic Acids Research

The Cockayne syndrome group B protein is a functional dimer

FEBS Journal

ATP-dependent chromatin remodeling by the Cockayne syndrome B DNA repair-transcription-coupling factor

Molecular and Cellular Biology

The biology of chromatin remodeling complexes

Annual Review of Biochemistry

Identical mutations in the CSB gene associated with either Cockayne syndrome or the DeSanctis-cacchione variant of xeroderma pigmentosum

Human Molecular Genetics

Alterations in the CSB gene in three Italian patients with the severe form of Cockayne syndrome (CS) but without clinical photosensitivity

Human Molecular Genetics

A quantitative atlas of mitotic phosphorylation

Proceedings of the National Academy of Sciences of the United States of America

AP-1: a double-edged sword in tumorigenesis

Nature Reviews Cancer

Noncovalent modification of chromatin: different remodeled products with different ATPase domains

Cold Spring Harbor Symposia on Quantitative Biology