Regulation of Murine Telomere Length by Rtel

117

149

We have investigated the DNA substrate specificity of BACH1 (BRCA1-associated C-terminal helicase). The importance of various DNA structural elements for efficient unwinding by purified recombinant BACH1 helicase was examined. The results indicated that BACH1 preferentially binds and unwinds a forked duplex substrate compared with a duplex flanked by only one single-stranded DNA (ssDNA) tail. In support of its DNA substrate preference, helicase sequestration studies revealed that BACH1 can be preferentially trapped by forked duplex molecules. BACH1 helicase requires a minimal 5 ssDNA tail of 15 nucleotides for unwinding of conventional duplex DNA substrates; however, the enzyme is able to catalytically release the third strand of the homologous recombination intermediate D-loop structure irrespective of DNA tail status. In contrast, BACH1 completely fails to unwind a synthetic Holliday junction structure. Moreover, BACH1 requires nucleic acid continuity in the 5 ssDNA tail of the forked duplex substrate within six nucleotides of the ssDNA-dsDNA junction to initiate efficiently DNA unwinding. These studies provide the first detailed information on the DNA substrate specificity of BACH1 helicase and provide insight to the types of DNA structures the enzyme is likely to act upon to perform its functions in DNA repair or recombination.Germ line mutations in BRCA1 lead to an increased lifetime risk of breast and/or ovarian cancer. Cellular studies have revealed that the BRCA1 tumor suppressor gene is required for the maintenance of genomic integrity and a normal level of resistance to DNA damage (for review see Refs. 1-3). The nuclear phosphoprotein BRCA1 contains tandem C-terminal BRCT motifs, a conserved protein sequence found in a large number of DNA damage-response proteins (4). The integrity of the BRCT motifs is required for the role of BRCA1 in double strand break repair (DSBR) 1 and homologous recombination (5-8). Tumor-predisposing missense and deletion mutations in the BRCA1 BRCT domain, all of which render BRCA1 defective in its DSBR function, also disrupt the ability of BRCA to bind BACH1. BACH1 is a member of the DEAH subfamily of superfamily 2 helicases (9). Consistent with its predicted helicase domain, BACH1 was recently shown to catalyze DNA unwinding of M13 partial duplex substrates and have a 5Ј to 3Ј polarity on a linearized M13 directionality substrate (10). A role of BACH1 helicase in DSBR was suggested by the observation that overexpression of a BACH1 allele (K52R) carrying a mutation in its ATP-binding pocket that inactivates its ATPase/ helicase function (10) resulted in a marked decrease in the ability of cells to repair DSBs, and that this dominant negative phenotype depended on a specific interaction between BACH1 and BRCA1 (9). More recently, it was shown that the interaction between BRCA1 and BACH1 depends on the phosphorylation status of BACH1 (11-13), and that this phosphorylationdependent interaction is required for DNA damage-induced checkpoint control during the G 2 /M phase of the ...

Section: Discussionmentioning

confidence: 99%

“…DNA Binding Assays-Protein/DNA binding mixtures (20 l) contained the indicated concentrations of BACH1 and 0.5 nM of the specified 32 P-end-labeled DNA substrate in the same reaction buffer as that used for helicase assays (see above) containing 2 mM ATP␥S (Fig. 1A).…”

Section: Methodsmentioning

confidence: 99%

Analysis of the DNA Substrate Specificity of the Human BACH1 HelicaseAssociated with BreastCancer

Gupta

Sharma

Sommers

et al. 2005

117

149

“…Interestingly, another DEXH helicase termed dog-1 from the nematode Caenorhabditis elegans appears important for maintenance of certain long guanine-rich DNA tracts and has been suggested to resolve G4-DNA arising on the parental lagging strand of DNA replication, although biochemical specificity of dog-1 activity awaits characterization (31). One mouse homologue of dog-1 termed Rtel (Regulator of telomere length) was found to induce loss of telomeres and a high frequency of chromosomal fusions when knocked out in mouse ES cells (32). If both Rtel and DHX36 protein helicases are indeed recognizing G4-DNA structures, certain DEXH proteins may represent a new family of DNA caretaker enzymes specializing in preventing quadruplex G4-DNA induced mutations.…”

Section: Discussionmentioning

confidence: 99%

The DEXH Protein Product of the DHX36 Gene Is the Major Source of Tetramolecular Quadruplex G4-DNA Resolving Activity in HeLa Cell Lysates

Vaughn

Creacy

Routh

et al. 2005

163

182

G4-DNA is a highly stable alternative DNA structure that can form spontaneously in guanine-rich regions of single-stranded DNA under physiological conditions. Since a number of biological processes create such single-stranded regions, G4-DNA occurrence must be regulated. To date, resolution of tetramolecular G4-DNA into single strands (G4-resolvase activity) has been observed only in recombinant RecQ DNA helicases. We previously reported that human cell lysates possess tetramolecular G4-DNA resolving activity (Harrington, C., Lan, Y., and Akman, S. (1997) J. Biol Chem. 272, 24631-24636). Here we report the first complete purification of a major non-RecQ, NTP-dependent G4-DNA resolving enzyme from human cell lysates. This enzyme is identified as the DEXH helicase product of gene DHX36 (also known as RHAU). G4-DNA resolving activity was captured from HeLa cell lysates on G4-DNA affinity beads and further purified by gel filtration chromatography. The DHX36 gene product was identified by mass spectrometric sequencing of a tryptic digest from the protein band on SDS-PAGE associated with activity. DHX36 was cloned within a His 6 -tagging vector, expressed, and purified from Escherichia coli. Inhibition and substrate resolution assays showed that recombinant DHX36 protein displayed robust, highly specific G4-DNA resolving activity. Immunodepletion of HeLa lysates by a monoclonal antibody to the DHX36 product removed ca. 77% of the enzyme from lysates and reduced G4-DNA resolving activity to 46.0 ؎ 0.4% of control, demonstrating that DHX36 protein is responsible for the majority of tetramolecular G4-DNA resolvase activity.G4-DNA is an alternative highly stable DNA structure forming within runs of guanine bases. It has been amply described previously (1). G4-DNA structures have the potential to disrupt normal duplex DNA; therefore, it might be expected that the genome would have minimized the usage of runs of deoxyguanosine. On the contrary, a growing body of data support the hypothesis that formation of G4-DNA in vivo is a recognized structural motif of specialized utility for key biological processes. Recent studies with a fluorescent G4-DNAbinding ligand, as well as a specific G4-DNA-binding protein, support the presence of G4-DNA structures located at human telomeres in vivo (2, 3). In addition to the aforementioned telomeres, other guanine-rich regions in human DNA readily form G4-DNA structures in vitro and make up specific genetic control elements, including the immunoglobin heavy chain switch region (4), guanine-rich regions of ribosomal DNA (5), the d(pCGG) repeats of the fragile X mental retardation gene (6), promoters of proliferation-associated genes, such as the c-MYC (7, 8), PDGF-A (9), RET (10), and the diabetes susceptibility locus IDDM2 promoter (11). It has been shown that a unimolecular G4-DNA structure has a repressor function in the c-MYC promoter (8). Compounds that stabilize G4-DNA in vivo have generated much interest because of their antitumor activity, suggesting that G4-DNA structures might be ...

“…In this regard G4R1/RHAU may be a candidate tumor suppressor gene, as the FANCJ DEXH helicase has proven to be in breast cancer (38). It will be of great interest to follow the replication of G-rich regions in cells down-regulated and knocked out for G4R1/RHAU and determine whether such G-rich sequences are preferentially lost over cell divisions, as was found for the Caenorhabditis elegans protein dog-1 and its mouse homolog Rtel (39,40). It is important to note that G4R1/RHAU is unique in being both an avid binder of quadruplex and a helicase without detectable nuclease activity.…”

mentioning

confidence: 99%

G4 Resolvase 1 Binds Both DNA and RNA Tetramolecular Quadruplex with High Affinity and Is the Major Source of Tetramolecular Quadruplex G4-DNA and G4-RNA Resolving Activity in HeLa Cell Lysates

Creacy

Routh

Iwamoto

et al. 2008

170

209

Quadruplex structures that result from stacking of guanine quartets in nucleic acids possess such thermodynamic stability that their resolution in vivo is likely to require specific recognition by specialized enzymes. We previously identified the major tetramolecular quadruplex DNA resolving activity in HeLa cell lysates as the gene product of DHX36 (Vaughn, J. P., Creacy, S. D., Routh, E. D., Joyner-Butt, C., Jenkins, G. S., Pauli, S., Nagamine, Y., and Akman, S. A. (2005) J. Biol Chem. 280, 38117-38120), naming the enzyme G4 Resolvase 1 (G4R1). G4R1 is also known as RHAU, an RNA helicase associated with the AU-rich sequence of mRNAs. We now show that G4R1/RHAU binds to and resolves tetramolecular RNA quadruplex as well as tetramolecular DNA quadruplex structures. The apparent K d values of G4R1/RHAU for tetramolecular RNA quadruplex and tetramolecular DNA quadruplex were exceptionally low: 39 ؎ 6 and 77 ؎ 6 pM, respectively, as measured by gel mobility shift assay. In competition studies tetramolecular RNA quadruplex structures inhibited tetramolecular DNA quadruplex structure resolution by G4R1/RHAU more efficiently than tetramolecular DNA quadruplex structures inhibited tetramolecular RNA quadruplex structure resolution. Down-regulation of G4R1/ RHAU in HeLa T-REx cells by doxycycline-inducible short hairpin RNA caused an 8-fold loss of RNA and DNA tetramolecular quadruplex resolution, consistent with G4R1/RHAU representing the major tetramolecular quadruplex helicase activity for both RNA and DNA structures in HeLa cells. This study demonstrates for the first time the RNA quadruplex resolving enzymatic activity associated with G4R1/RHAU and its exceptional binding affinity, suggesting a potential novel role for G4R1/ RHAU in targeting in vivo RNA quadruplex structures.The nucleobase guanine is unique among bases in nucleic acids because of its great tendency toward self-association. Stable cyclic guanine quartets form by self-assembly through Hoogsteen hydrogen bonding between each of the four guanine molecules (N 1 -H and N 2 -H share hydrogen with O 6 and N 7 of the adjacent guanine). In the presence of physiological monovalent cations these quartets can self-associate into vertical stacks that coordinately bond a cation at the center of the G quartets, and the assemblies are further stabilized through stacking interactions (reviewed in Ref. 1). This vertical assembly of G quartets is known as G-quadruplex; this self-associating phenomenon is responsible for the observation that over 30 different derivatives of guanine can form gels in water (2). These are also the forces that stabilize the formation of quadruplex structures in nucleic acids that are also termed G4-DNA and G4-RNA. Since the initial observation by Sen and Gilbert (3) of the formation of tetramolecular G4-DNA, it has been shown that DNA or RNA molecules possessing runs of as few as three guanines in a row can form a variety of extraordinarily stable quadruplex structures (reviewed in Ref. 4). In fact, guanine quadruplex structures create th...