Biochimica et Biophysica Acta 1822 (2012) 1727–1734 Contents lists available at SciVerse ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbadis RAPADILINO RECQL4 mutant protein lacks helicase and ATPase activity Deborah L. Croteau, Marie L. Rossi, Jennifer Ross, Lale Dawut, Christopher Dunn, Tomasz Kulikowicz, Vilhelm A. Bohr ⁎ Laboratory of Molecular Gerontology, National Institute on Aging, Baltimore, MD, 21224, USA article info abstract Article history: The RecQ family of helicases has been shown to play an important role in maintaining genomic stability. In Received 18 May 2012 humans, this family has five members and mutations in three of these helicases, BLM, WRN and RECQL4, are Received in revised form 17 July 2012 associated with disease. Alterations in RECQL4 are associated with three diseases, Rothmund–Thomson Accepted 26 July 2012 syndrome, Baller–Gerold syndrome, and RAPADILINO syndrome. One of the more common mutations found in Available online 31 July 2012 RECQL4 is the RAPADILINO mutation, c.1390+2delT which is a splice-site mutation leading to an in-frame skip- ping of exon 7 resulting in 44 amino acids being deleted from the protein (p.Ala420–Ala463del). In order to char- Keywords: fi RecQ helicase acterize the RAPADILINO RECQL4 mutant protein, it was expressed in bacteria and puri ed using an established RECQL4 protocol. Strand annealing, helicase, and ATPase assays were conducted to characterize the protein's activities RAPADILINO relative to WT RECQL4. Here we show that strand annealing activity in the absence of ATP is unchanged from Rothmund–Thomson syndrome that of WT RECQL4. However, the RAPADILINO protein variant lacks helicase and ssDNA-stimulated ATPase ATPase activity. These observations help explain the underlying molecular etiology of the disease and our findings provide insight into the genotype and phenotype association among RECQL4 syndromes. Published by Elsevier B.V. 1. Introduction of the distinguishing features of RTS and BGS but is lacking in RAPADILINO. Another feature found in RTS patients but missing in The RecQ helicase family of proteins is critically important for RAPADILINO patients is alopecia, which includes deficiencies in hair genome maintenance and in human cells there are five RecQ family ho- growth including the absence of eyelashes and eyebrows. More recently mologues: RECQL1, WRN, BLM, RECQL4 and RECQL5 (for recent reviews it has been recognized that RAPADILINO and type II RTS patients share see Refs. [1,2]). Mutations in WRN or BLM genes give rise to Werner a common predisposition for osteosarcomas and lymphomas [4].An (OMIM ID 277700) or Bloom syndromes (OMIM ID 210900), respec- elevated risk for osteosarcoma is also a characteristic feature seen in tively, whereas mutations within RECQL4 give rise to three related WRN syndrome patients [7]. While the three RECQL4-associated syn- but distinct diseases: RAPADILINO (OMIM ID 266280), Rothmund– dromes share some common features, their unique spectrum of clinical Thomson (RTS, OMIM ID 268400), and Baller–Gerold (BGS, OMIM ID manifestations highlights the complexity of deciphering the clinical 218600) syndromes. Not all reported RTS patients have identifiable phenotypes associated with patient RECQL4 genotypes. mutations within the RECQL4 gene, and therefore, there are two classes The RECQL4 gene encompasses 21 exons, and exons 8–14 encode the of RTS patients: type I which lack RECQL4 mutations and type II which conserved RecQ helicase domain. The most common RECQL4 mutation have confirmed RECQL4 mutations [3].Atthistimeitisnotclearwhy in RAPADILINO patients is the c.1390+2delT (p.Ala420–Ala463del) mutations in the RECQL4 gene gives rise to three different clinical [4,8]. This mutation is found in the majority of reported RAPADILINO syndromes. patients, especially those of Finnish decent, and importantly, it is one All three RECQL4-associated diseases are rare, autosomal recessive of a few homozygous RECQL4 mutations. The mutation destroys a splice and characterized by growth retardation and bone malformations, es- acceptor site resulting in skipping of exon 7, which immediately pecially radial ray defects (for recent reviews see Refs. [4,5]). The precedes the exons responsible for encoding the helicase domain of RAPADILINO syndrome was first identified in 1989, and the name is RECQL4. Therefore, loss of this exon could alter the protein's function. an acronym for the features observed in patients: radial hypoplasia/ The region of RECQL4 encoded by exon 7 has been previously reported aplasia, patellar hypoplasia/aplasia, cleft or highly arched palate, diar- to be important for nuclear import and retention, because the GFP rhea and dislocated joints, little size and limb malformation, and slender fusion of RECQL4 protein harboring the major RAPADILINO patient nose and normal intelligence [6]. Poikiloderma, a cutaneous rash, is one mutation (RAPA) was overwhelmingly mislocalized to the cytoplasm [9]. More recently, our laboratory has shown that this region of RECQL4 is also important for localization to focal laser-induced DNA damage ⁎ Corresponding author at: NIH Biomedical Research Center, 251 Bayview Boulevard, Baltimore, MD, 21224, USA . Tel.: +1 4105588162. [10]. In the literature, the major RAPA mutation is thought to spare the E-mail address: [email protected] (V.A. Bohr). helicase domain [8]; however there is no published information about 0925-4439/$ – see front matter. Published by Elsevier B.V. http://dx.doi.org/10.1016/j.bbadis.2012.07.014 1728 D.L. Croteau et al. / Biochimica et Biophysica Acta 1822 (2012) 1727–1734 whether this is true or if this mutation alters the biochemical activities of with sites of DNA replication and DNA replication initiation proteins the RECQL4 protein. [23–25]. RECQL4 is the least well characterized protein among the mammalian Here we have characterized the biochemical properties of the major RecQ helicases, whose deficiencies cause human disease. RecQ helicase RAPA patient mutant protein, RAPA RECQL4. This mutation is a RECQL4 proteins typically share three structural elements: the conserved helicase gene mutation found in the homozygous state in patients. The mutation domain, a RecQ helicase conserved domain (RQC) and a helicase is the result of a splice-site mutation resulting in the in-frame skipping and RNase D C-terminal domain (HRDC). RECQL4 is unique among the of exon 7. Consequently, the RAPA RECQL4 protein lacks 44 amino acids mammalian proteins because it lacks the RQC and HRDC domains. In just prior to the helicase domain. The WT and RAPA RECQL4 recombi- the other RecQ helicase proteins, these two domains have been shown nant proteins were purified from bacteria and analyzed in vitro for to be important for DNA binding and for mediating protein:protein inter- strand annealing, helicase, and ssDNA-stimulated ATPase activities. actions (reviewed in Refs. [1,2]). In the original characterization of Our analysis showed that the RAPA patient mutation compromised RECQL4, it was described as lacking helicase activity [11].However, both the helicase and ATPase activities of the protein while sparing more recent work has demonstrated that RECQL4 in fact does possess the strand annealing activity. Thus, not only does the RAPA mutation helicase activity, although with a very limited substrate specificity cause mislocalization of the RECQL4 protein, it also severely impairs [12–14]. It was proposed that the robust strand annealing activity of the protein's catalytic functions. Thorough biochemical characterization RECQL4 was responsible for masking the helicase activity and only of RECQL4 patient mutations may contribute to a better understanding upon addition of excess single stranded DNA was the activity revealed of the genotype:phenotype relationships between RECQL4 mutations [12]. Upon further characterization, our laboratory has shown that by and the three RECQL4-associated syndromes RAPADILINO, RTS and employing short forked DNA substrates RECQL4's inherent helicase BGS. Only through such analysis are we likely to unravel how RECQL4 activity can be readily observed without the need for excess single mutations contribute to the disease phenotypes. stranded DNA [14]. Thus, like other RecQ helicases, RECQL4 possesses multiple biochemical functions including: DNA binding, DNA strand 2. Results annealing, the classic 3′ to 5′ helicase and DNA-stimulated ATPase activ- ities [11–14]. 2.1. Production of RAPA RECQL4 The in vivo biochemical functions and pathways in which RECQL4 plays a role are not yet fully characterized. The protein has been shown RECQL4 is found on chromosome 8q, and its genomic organization to localize to multiple cellular compartments such as the nucleus, nucle- is comprised of 21 exons across 6.5 kb of DNA with a high number of olus, cytoplasm and mitochondria [15–19]. The precise localization of unusually short intervening introns [26]. The intron-exon boundaries RECQL4 is cell type specific as, some cells show more cytoplasmic than of WT and RAPA RECQL4 are shown in Fig. 1A. The N-terminus of the nuclear localization [15]. Acetylation by p300 may also contribute to the protein shares sequence similarity to Sld2/DRC1, a yeast protein differential localization patterns of RECQL4 [20].Additionally,RECQL4 involved in DNA replication [23]. In the middle of the protein, exons has been shown to localize to sites experiencing DNA damage including 8–14 encode the conserved helicase domain. The RAPA mutation, foci found following oxidatively generated DNA damage and double c.1390+2delT, causes deletion of exon 7 from RECQL4's mRNA, but strand break DNA damage [10,16,21,22]. There is evidence that RECQL4 it does not result in the introduction of a premature termination modulates base excision DNA repair [21] and that it plays a role in DNA codon; thus the protein coded by this mRNA variant lacks 44 amino double strand break repair [10,16]. However, its role in DNA repair is acids just upstream from the conserved helicase domain. much less clear than the roles of the other human disease-causing RecQ The WT and RAPA RECQL4 proteins were expressed and purified helicases (WRN and BLM).