Biochemical characterization of the RECQ4 protein, mutated in Rothmund-Thomson syndrome
Introduction
DNA helicases play essential roles in DNA metabolism, including replication, transcription, recombination, and repair. The RecQ helicase family has been implicated in double-strand break repair and homologous recombination and is important for the maintenance of genomic integrity [1]. Members of this protein family have been identified from E. coli to humans [1]. While yeast and bacteria possess a single RecQ helicase, there are five apparent orthologs in humans. Mutations in three human RecQ-like helicases, BLM (RECQ2), WRN (RECQ3), and RECQ4 have been associated with Bloom's syndrome (BS), Werner's syndrome (WS), and Rothmund-Thomson syndrome (RTS), respectively. Patients with all three of these disorders show increased cancer susceptibility and genomic instability [2], [3], although the clinical features of the disorders and spectrum of cancers observed in the patients are different.
RTS is an autosomal recessive disorder characterized by growth deficiency, juvenile cataracts, skin hyperpigmentation, poikiloderma, brittle hair, photosensitivity, congenital skeletal defects, and a predisposition to malignancy, especially osteosarcomas [4], [5], [6]. Over half of patients with RTS harbor mutations in the RECQ4 gene [7], [8], [9], [10], [11]. The RECQ4 gene encodes a 1208 amino acid protein with a predicted molecular weight of 133 kDa [12], [13] and contains sequence features that define the RecQ family [1]. Most of the identified mutations in the RTS patients are within the conserved helicase domain of RECQ4 spanning exons 8–14 [7], [8], [9], and these mutations are predicted to produce proteins truncated in this domain. Aside from the helicase domain, RECQ4 lacks significant homology to other known proteins. The RECQ4 protein is normally present in the nucleus, but in certain transformed cells, it becomes overexpressed and a majority of it is found in the cytoplasm, where it seems to associate with the ubiquitin ligases UBR1 and UBR2 [14]. The biological role of the RECQ4/UBR1/UBR2 complex is currently unknown.
Disorders associated with RecQ helicase deficiency, including BS and WS, show genomic instability at the cellular level as well as hypersensitivity to DNA-damaging agents, such as topoisomerase inhibitors and DNA cross-linking agents [15]. BS cells also have a highly elevated frequency of sister chromatid exchanges (SCEs) [16], implicating the BLM protein in the prevention of this type of genetic exchange. Although elevated levels of SCEs have not been reported for RTS cells, high frequencies of chromosomal instability, including trisomies and isochromosomes, have been observed [8], [17], [18], [19].
Consistent with the chromosomal instability observed in the RTS patients, cells derived from a RECQ4 mutant mouse show high frequencies of aneuploidy and appear to have defects in sister chromatid cohesion [20], suggesting that RECQ4 has a role in regulating this process. In both yeast and mammalian cells, cohesin is recruited to chromosomes specifically in response to DNA damage [21], [22]. In fact, Mre11 and Rad50, components of the homologous recombination pathway, are required for accumulation of cohesin near the site of a DSB [21], [23]. It remains to be determined whether RECQ4 has a role in DSB repair by HR, as has been proposed for other members of the RecQ helicase family [1].
Biochemical evidence supports a role for the RecQ helicases in the processing of aberrant DNA structures that arise during DNA replication and repair. BLM, WRN, RECQ1, RECQ5β (the largest RECQ5 isoform that results from alternative splicing of the RECQ5 transcript [24]), Saccharomyces cerevisiae Sgs1, and E. coli RecQ all possess a DNA-dependent ATPase activity and a 3′ to 5′ DNA helicase activity that is fueled by ATP hydrolysis [1]. Although their substrate specificities are not identical, these helicases can unwind a variety of potentially recombinogenic DNA structures, including four-way junctions and D-loops [25], [26], [27], [28], [29]. In addition to its DNA helicase activity, RECQ5β possesses a single-strand DNA annealing activity that is inhibited by the single-strand binding protein, RPA [29].
In order to decipher the mechanistic role that RECQ4 plays in biological pathways germane for genome maintenance, it is necessary to purify this protein and characterize it biochemically. Here, we describe the initial biochemical characterization of the human RECQ4 protein expressed in and purified to near homogeneity from E. coli. We show that RECQ4 has a ssDNA-dependent ATPase activity and a DNA strand-annealing activity. Unlike the previously characterized members of the RecQ family, RECQ4 lacks a detectable DNA helicase activity.
Section snippets
RecQ4 expression and purification
The RECQ4 gene was amplified by PCR from a plasmid containing the RECQ4 cDNA using primers RECQ4-EcoRI (CCGGAATTCATGGAGCGGCTGCGGGACGTGCG) and RECQ4(His)-XhoI (CCGCTCGAGTTAGTGATGGTGATGGTGATGGTGATGGTGCCCAGGGCGGGCCACCTGCAGGAGCTCT) and cloned into the EcoRI/XhoI sites of pGEX-6P1 (Amersham Biosciences). The resulting plasmid pGEX::RecQ4-HIS9 containing the RECQ4 gene with a cleavable N-terminal glutathione S-transferase (GST) tag and a C-terminal 9-histidine tag under the control of the IPTG
RecQ4 purification
To facilitate the purification of RECQ4, GST and 9-histidine tags were added to its amino-terminus and carboxyl-terminus, respectively. Even though the expression level of full length GST-RECQ4-His9 was difficult to detect by Coomassie Blue staining of SDS-PAGE gels in which cell extracts had been run, we were able to verify protein expression by immunoblot analysis using anti-GST antibodies. We found that RECQ4 is quite susceptible to intracellular proteolysis in bacteria, yielding several
Discussion
In summary, we have successfully expressed full-length human RECQ4 protein in E. coli, purified it to near homogeneity, and carried out its initial biochemical characterization. Our studies have revealed a ssDNA-dependent ATPase activity in RECQ4, which is a characteristic shared by other members of the RecQ protein family [1]. We have also determined various parameters that influence the RECQ4 ATPase activity. We note that an immunoprecipitate containing RECQ4 and associated factors obtained
Acknowledgments
This work was supported by National Institutes of Health grants RO1CA110415 and RO1CA96953, as well as Department of Defense Grant DAMD17-03-1-0586. We are grateful to Rohit Prakash for his help in generating the helicase substrates. We also thank Dr. Michelle Duquette for her advice in forming G4 DNA, and we thank Dr. Nicholas Sherman for the MS analysis.
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Note: As of August 1, 2005, Dr. Lumir Krejci can be contacted at National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kotlarska 2, Brno 611 37, Czech Republic.