RecQ4 is a member of the RecQ helicase family, an evolutionarily conserved class of enzymes, dedicated to preserving genomic integrity by operating in telomere maintenance, DNA repair and ...replication. While reduced RecQ4 activity is associated with cancer predisposition and premature aging, RecQ4 upregulation is related to carcinogenesis and metastasis. Within the RecQ family, RecQ4 assumes an exceptional position, lacking several characteristic RecQ domains. Here we present the crystal structure of human RecQ4, encompassing the conserved ATPase core and a novel C-terminal domain that lacks resemblance to the RQC domain observed in other RecQ helicases. The new domain features a zinc-binding site and two distinct types of winged-helix domains, which are not involved in canonical DNA binding or helicase activity. Based on our structural and functional analysis, we propose that RecQ4 exerts a helicase mechanism, which may be more closely related to bacterial RecQ helicases than to its human family members.
Ctf18-RFC is an alternative PCNA loader which plays important but poorly understood roles in multiple DNA replication-associated processes. To fulfill its specialist roles, the Ctf18-RFC clamp loader ...contains a unique module in which the Dcc1-Ctf8 complex is bound to the C terminus of Ctf18 (the Ctf18-1-8 module). Here, we report the structural and functional characterization of the heterotetrameric complex formed between Ctf18-1-8 and a 63 kDa fragment of DNA polymerase ɛ. Our data reveal that Ctf18-1-8 binds stably to the polymerase and far from its other functional sites, suggesting that Ctf18-RFC could be associated with Pol ɛ throughout normal replication as the leading strand clamp loader. We also show that Pol ɛ and double-stranded DNA compete to bind the same winged-helix domain on Dcc1, with Pol ɛ being the preferred binding partner, thus suggesting that there are two alternative pathways to recruit Ctf18-RFC to sites of replication.
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•Crystal structure of a eukaryotic leading strand polymerase:clamp loader complex•Ctf18-RFC may be associated with the leading strand throughout normal replication•A winged-helix domain on Ctf18-RFC recognizes two distinct substrates•DNA Pol ɛ outcompetes dsDNA for recruiting Ctf18-RFC to the replisome
Grabarczyk et al. structurally and functionally characterize the interaction between the yeast leading strand DNA polymerase ɛ and the alternative clamp loader Ctf18-RFC. Their study reveals that Ctf18-RFC may associate with Pol ɛ during normal replication through an unusual bifunctional winged-helix domain.
XPD, as part of the TFIIH complex, has classically been linked to the damage verification step of nucleotide excision repair (NER). However, recent data indicate that XPD, due to its iron-sulfur ...center interacts with the iron sulfur cluster assembly proteins, and may interact with other proteins in the cell to mediate a diverse set of biological functions including cell cycle regulation, mitosis, and mitochondrial function. In this perspective, after first reviewing the function and some of the key disease causing variants that affect XPD’s interaction with TFIIH and the CDK-activating kinase complex (CAK), we investigate these intriguing cellular roles of XPD and highlight important unanswered questions that provide a fertile ground for further scientific exploration.
Abstract
Over the last decades the phase problem in macromolecular x-ray crystallography has become more controllable as methods and approaches have diversified and improved. However, solving the ...phase problem is still one of the biggest obstacles on the way of successfully determining a crystal structure. To overcome this caveat, we have utilized the anomalous scattering properties of the heavy alkali metal cesium. We investigated the introduction of cesium in form of cesium chloride during the three major steps of protein treatment in crystallography: purification, crystallization, and cryo-protection. We derived a step-wise procedure encompassing a “quick-soak”-only approach and a combined approach of CsCl supplement during purification and cryo-protection. This procedure was successfully applied on two different proteins: (i) Lysozyme and (ii) as a proof of principle, a construct consisting of the PH domain of the TFIIH subunit p62 from
Chaetomium thermophilum
for de novo structure determination. Usage of CsCl thus provides a versatile, general, easy to use, and low cost phasing strategy.
The XPD protein is a vital subunit of the general transcription factor TFIIH which is not only involved in transcription but is also an essential component of the eukaryotic nucleotide excision DNA ...repair (NER) pathway. XPD is a superfamily‐2 5′‐3′ helicase containing an iron–sulphur cluster. Its helicase activity is indispensable for NER and it plays a role in the damage verification process. Here, we report the first structure of XPD from Thermoplasma acidophilum (taXPD) in complex with a short DNA fragment, thus revealing the polarity of the translocated strand and providing insights into how the enzyme achieves its 5′‐3′ directionality. Accompanied by a detailed mutational and biochemical analysis of taXPD, we define the path of the translocated DNA strand through the protein and identify amino acids that are critical for protein function.
The first DNA‐bound structure of an SF2B‐type helicase reveals the DNA strand translocation polarity governing 5′‐3′ directionality in this important helicase family that also includes major genome stability regulators FANCJ and RTEL.
Damage recognition in nucleotide excision DNA repair Kuper, Jochen; Kisker, Caroline
Current opinion in structural biology,
February 2012, 2012-Feb, 2012-2-00, 20120201, Letnik:
22, Številka:
1
Journal Article
Recenzirano
► The β-hairpin is introduced as a general tool for damage detection in NER. ► UvrA uses an active mechanism for damage scanning. ► The iron sulphur cluster of XPD is linked to damage detection.
...Nucleotide excision repair (NER) is a highly versatile DNA repair process. Its ability to repair a large number of different damages with the same subset of recognition factors requires structural tools for damage recognition that are both broad and very accurate. Over the past few years detailed structural information on damage recognition factors from eukaryotic and prokaryotic NER has emerged. These structures shed light on the toolkit utilized in the damage recognition process and help explain the broad substrate specificity of NER.
Enhanced expression of the MYC transcription factor is observed in the majority of tumors. Two seemingly conflicting models have been proposed for its function: one proposes that MYC enhances ...expression of all genes, while the other model suggests gene-specific regulation. Here, we have explored the hypothesis that specific gene expression profiles arise since promoters differ in affinity for MYC and high-affinity promoters are fully occupied by physiological levels of MYC. We determined cellular MYC levels and used RNA- and ChIP-sequencing to correlate promoter occupancy with gene expression at different concentrations of MYC. Mathematical modeling showed that binding affinities for interactions of MYC with DNA and with core promoter-bound factors, such as WDR5, are sufficient to explain promoter occupancies observed in vivo. Importantly, promoter affinity stratifies different biological processes that are regulated by MYC, explaining why tumor-specific MYC levels induce specific gene expression programs and alter defined biological properties of cells.
The XPD helicase is a central component of the general transcription factor TFIIH which plays major roles in transcription and nucleotide excision repair (NER). Here we present the high-resolution ...crystal structure of the Arch domain of XPD with its interaction partner MAT1, a central component of the CDK activating kinase complex. The analysis of the interface led to the identification of amino acid residues that are crucial for the MAT1-XPD interaction. More importantly, mutagenesis of the Arch domain revealed that these residues are essential for the regulation of (i) NER activity by either impairing XPD helicase activity or the interaction of XPD with XPG; (ii) the phosphorylation of the RNA polymerase II and RNA synthesis. Our results reveal how MAT1 shields these functionally important residues thereby providing insights into how XPD is regulated by MAT1 and defining the Arch domain as a major mechanistic player within the XPD scaffold.
The eukaryotic XPD helicase is an essential subunit of TFIIH involved in both transcription and nucleotide excision repair (NER). Mutations in human XPD are associated with several inherited diseases ...such as xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. We performed a comparative analysis of XPD from Homo sapiens and Chaetomium thermophilum (a closely related thermostable fungal orthologue) to decipher the different molecular prerequisites necessary for either transcription or DNA repair. In vitro and in vivo assays demonstrate that mutations in the 4Fe4S cluster domain of XPD abrogate the NER function of TFIIH and do not affect its transcriptional activity. We show that the p44-dependent activation of XPD is promoted by the stimulation of its ATPase activity. Furthermore, we clearly demonstrate that XPD requires DNA binding, ATPase, and helicase activity to function in NER. In contrast, these enzymatic properties are dispensable for transcription initiation. XPD helicase is thus exclusively devoted to NER and merely acts as a structural scaffold to maintain TFIIH integrity during transcription.
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