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Mol Cell Biol. 2010 May; 30(10): 2460–2472.
Published online 2010 March 15. doi:  10.1128/MCB.01583-09
PMCID: PMC2863711

RecQL5 Promotes Genome Stabilization through Two Parallel Mechanisms—Interacting with RNA Polymerase II and Acting as a Helicase[down-pointing small open triangle]

Abstract

The RecQL5 helicase is essential for maintaining genome stability and reducing cancer risk. To elucidate its mechanism of action, we purified a RecQL5-associated complex and identified its major component as RNA polymerase II (Pol II). Bioinformatics and structural modeling-guided mutagenesis revealed two conserved regions in RecQL5 as KIX and SRI domains, already known in transcriptional regulators for Pol II. The RecQL5-KIX domain binds both initiation (Pol IIa) and elongation (Pol IIo) forms of the polymerase, whereas the RecQL5-SRI domain interacts only with the elongation form. Fully functional RecQL5 requires both helicase activity and associations with the initiation polymerase, because mutants lacking either activity are partially defective in the suppression of sister chromatid exchange and resistance to camptothecin-induced DNA damage, and mutants lacking both activities are completely defective. We propose that RecQL5 promotes genome stabilization through two parallel mechanisms: by participation in homologous recombination-dependent DNA repair as a RecQ helicase and by regulating the initiation of Pol II to reduce transcription-associated replication impairment and recombination.

RecQ helicases perform essential roles in maintaining genome stability (5). Mammals have five RecQ homologs: RecQL1, BLM/RecQL2, WRN/RecQL3, RecQL4, and RecQL5 (6, 12). Mutations in BLM, WRN, and RecQL4 give rise to the genomic instability disorders Bloom's syndrome, Werner's syndrome, and Rothmund-Thomson's syndrome, respectively. These disorders are characterized by cancer predisposition, chromosomal instability, and cellular hypersensitivity to DNA-damaging agents. Although RecQL5 has not been associated with any human disease, RecQL5/ mice exhibit an increased incidence of cancer, a phenotype common to all RecQ helicase syndromes (5, 16, 20).

RecQL5 may play a role in the stabilization and/or restart of stalled replication forks. This was suggested by findings that mouse RecQL5/ embryonic stem (ES) cells and primary embryonic fibroblasts are hypersensitive to camptothecin (CPT), a topoisomerase I inhibitor that blocks DNA replication (18, 19). In addition, RecQL5 may suppress homologous recombination (HR) and/or crossover events, as evidenced by the observation that mouse RecQL5/ cells display an elevated frequency of sister chromatid exchange (SCE) (18, 19). The roles of RecQL5 in the suppression of SCE can be replaced functionally by BLM in chicken DT40 cells, because the deletion of RecQL5 in normal DT40 cells does not lead to an elevated SCE frequency, whereas the deletion of RecQL5 in BLM/ cells results in a further increase of the SCE frequency that is higher than that of BLM/ cells (41).

RecQL5 possesses a DNA helicase activity similar to that of BLM, which may explain their overlapping roles in SCE suppression. Both helicases have 3′-to-5′ polarity and can promote branch migration for Holliday junctions (15), the displacement of D loops, and the disruption of Rad51 presynaptic filaments (20). However, RecQL5 cannot stimulate the dissolution of double Holliday junctions (20), a hallmark reaction for BLM (35, 43), suggesting that RecQL5 cannot substitute BLM for the suppression of crossover recombination. Indeed, although an elevated SCE level was not detected in RecQL5/ DT40 cells, it was observed in BLM/ cells, indicating that two proteins have both overlapping and nonoverlapping functions.

RecQL5 was previously shown to associate with a number of DNA-processing proteins, including Rad51 (20), topoisomerase 3α (Topo3α) and Topo3β (39), proliferating cell nuclear antigen (PCNA) (22), the Mre11-Rad50-Nbs1 (MRN) complex (47), and RNA polymerase II (Pol II) (3, 21). In vitro transcription assays and small interfering RNA (siRNA) studies have shown that the RecQL5-Pol II interaction inhibits transcriptional initiation and elongation (3, 4, 21). However, the mechanism of RecQL5 in promoting genome stabilization remains unclear due to a lack of a suitable cell-based system to assess the importance of various RecQL5 activities. Moreover, the domains in RecQL5 that are responsible for its interactions with its various partners have remained unknown.

In this study, we performed structural modeling and mutagenesis to identify two conserved domains in RecQL5 that interact with different forms of Pol II. We developed a DT40 cell-based system to show that RecQL5 protects genome stability through two parallel mechanisms—helicase action and interaction with the initiation form of Pol II.

MATERIALS AND METHODS

Cell culture.

Chicken DT40 cell lines were maintained in RPMI medium (Life Technology) supplemented with 10% heat-inactivated fetal calf serum, 1% chicken serum, 1.5% penicillin-streptomycin (Invitrogen), and 10 mM HEPES (pH 7.9) and were grown in a humidified carbon dioxide (CO2)-containing atmosphere at 39.5°C. HeLa S3 cells were obtained from the National Cell Culture Center.

Preparation of antibodies and plasmids.

A rabbit RecQL5 polyclonal antibody was raised against a chimeric protein containing a region of RecQL5 (amino acids [aa] 927 to 991) fused to maltose-binding protein. This antibody was affinity purified by using the immunogen as the matrix. The antibody works only for immunoblotting analysis but not for immunoprecipitation. Polyclonal antibodies against BLM, Topo 3α, and Topo 3β were described elsewhere previously (29, 42). Rad51 (H-92) and PCNA (PC-10) antibodies were obtained from Santa Cruz Biotechnology, anti-Flag M2 monoclonal antibody was obtained from Sigma, anti-MRN complex antibodies were obtained from BD Transduction Laboratories, and Pol II antibodies 8WG16 and ARNA-3 were obtained from Upstate and Fitzgerald, respectively.

Expression vectors of Flag-tagged full-length RecQL5 and deletion mutants were constructed according to standard molecular biology techniques. The PCR products were digested with appropriate restriction enzymes and cloned into the corresponding restriction sites of the expression vector pIRESneo3 (Clontech Laboratories, Inc.). Site-directed mutagenesis was carried out with a QuikChange multisite-directed mutagenesis kit according to the manufacturer's protocol (Stratagene).

Immunoprecipitation.

The nuclear and cytoplasmic extracts were prepared as described previously (46). Briefly, the nuclear extract was prepared by extracting the nuclear pellet twice with buffer C containing 20 mM HEPES (pH 7.9), 0.42 M NaCl, 25% glycerol, 1.5 mM MgCl2, and 0.2 mM EDTA.

RecQL5-associated complexes were isolated from nuclear extracts of HeLa cells stably expressing Flag-tagged RecQL5 (FRecQL5). Flag immunoprecipitation was done according to the manufacturer's protocol (Sigma). In brief, 1 ml (8 mg/ml) of nuclear extract was diluted five times with immunoprecipitation (IP) buffer (20 mM HEPES [pH 7.9], 200 mM NaCl, 1 mM dithiothreitol [DTT], 0.2 mM phenylmethylsulfonyl fluoride [PMSF], 10% glycerol) and incubated with anti-Flag M2 agarose affinity gel (Sigma) for at least 12 h at 4°C. The immunoprecipitate on the M2 beads was washed three times for 10 min each in IP buffer and eluted with the Flag peptide (300 ng/ml) in phosphate-buffered saline. The eluted complex was subjected to silver staining and immunoblotting. For mass spectrometric analysis, the proteins were visualized by silver staining, excised from the gel, and digested with trypsin. The peptides obtained were analyzed by matrix-assisted laser desorption ionization-time of flight and/or liquid chromatography mass spectrometry (MS) analysis. The data are not shown but are available upon request. Pol II was similarly immunoprecipitated from HeLa nuclear extracts and analyzed by mass spectrometry and immunoblotting.

For immunodetection of other known RecQL5-interacting partners (RAD51, PCNA, and the MRN complex), Flag-tagged RecQL5 was immunoprecipitated by use of a previously described protocol (21) and analyzed by immunoblotting. In this case, immunoprecipitation from total cell lysates was performed in lysis buffer (50 mM Tris-HCl [pH 7.5], 0.5 mM EDTA, 1.0% Triton X-100, and 200 mM NaCl) in the presence of a protease inhibitor cocktail (Roche Molecular Biochemicals).

Complementation analyses.

FRecQL5 was cloned into the pCDNA3.1/Zeo+ expression vector (Invitrogen Life Technologies). FRecQL5 point mutants were generated by using a QuikChange multisite-directed mutagenesis kit (Stratagene). The resulting constructs were transfected into BLM//RecQL5/ chicken DT40 cells by using nucleofection solution according to the manufacturer's protocol (Amaxa). Briefly, 2 × 106 cells in pellets were suspended into 100 μl of nucleofection solution, and 2 μg of DNA was then added to the suspended solutions. Due to the low transfection efficiency, each construct was digested with XhoI, and the linearized plasmids were used for transfection. Transfected solutions were incubated for 24 h in a six-well plate with 1.5 ml of medium. Stable clones were isolated from the primary transfectant pools using limiting dilution into 96-well plates and selection for resistance to zeocin (Invitrogen).

SCE assay.

SCE staining was performed as described previously (33). Cultures were grown through two cell cycles to achieve the preferential labeling of sister chromatid in the presence of 10 μM 5-bromodeoxyuridine. Colcemid (Sigma) was added at a final concentration of 0.1 μg/ml to accumulate mitotic cells 2 h prior to the harvesting of cells. Harvested cells were then treated with 75 mM KCl (hypotonic solution) for 25 min at room temperature and then fixed with 3:1 (vol/vol) methanol-glacial acetic acid. The fixed cell suspension was dropped onto a glass slide and air dried. The cells on the slides were incubated with 10 μg of Hoechst 33258/ml in 50 mM phosphate buffer (pH 6.8) for 20 min and rinsed with MacIlvaine solution (164 mM Na2HPO4, 16 mM citric acid [pH 7.0]). The cells were exposed to UV at a distance of 1 cm for 60 min and then incubated in 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) at 58°C for 20 min. The cells were finally stained with 3% Giemsa solution dissolving in 50 mM phosphate buffer at pH 6.8 for 25 min and examined under a light microscope.

Measurement of cellular sensitivity to CPT.

Growth inhibition experiments with CPT were carried out with 96-well flat-bottomed microplates, and the amount of viable cells at the end of the incubation period was determined by a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay according to the manufacturer's protocols (Promega Corporation). Thus, 5,000 cells/well in 50 μl were plated, and another 50 μl of the drug or medium alone was added as a control. The cells were cultured for 3 days. After the addition of MTS (10 μl/well), the plates were incubated for an additional 2 h. The absorbance was measured at 490 nm by using a 1420 multilabel counter (Victor3V; Perkin-Elmer).

Homology modeling.

Homology models were generated by using the alignments described below and the Swiss Model alignment server (2). Models were evaluated by using the MODFOLD and QMEAN servers, and comparisons of composite residue error scores were made between the template and the model (7, 28).

RESULTS

Pol II is a major component of a RecQL5 complex.

To purify the RecQL5 (referred to as the RecQL5β isoform)-associated complex, we generated a HeLa cell line stably expressing Flag-tagged RecQL5 (FRecQL5). Flag immunoprecipitation (IP) of FRecQL5 followed by SDS-PAGE revealed the presence of three major polypeptides (Fig. (Fig.1a,1a, compare lanes 2 and 3). The polypeptides were later determined by mass spectrometry (MS) to be RecQL5 and the two largest subunits of Pol II, RPB1 and RPB2. The fact that RPB1 and RPB2 are the major polypeptides in the FRecQL5 IP suggests that Pol II is a main component of a RecQL5-associated complex. This notion is further supported by the following evidence. First, immunoblotting confirmed the presence of Pol II in the FRecQL5 IP (Fig. (Fig.1b,1b, lane 6). Second, MS analyses of the entire FRecQL5 IP mixture detected the presence of six additional subunits of Pol II (RBP3, -4, -5, -8, -9, and -11) (see Fig. S1a in the supplemental material). Third, we performed reciprocal IP with a Pol II antibody using HeLa cells expressing FRecQL5 and identified RecQL5 as a major polypeptide of Pol II by silver staining and MS analyses (Fig. (Fig.1c,1c, lane 2, and see Fig. S1b in the supplemental material). Fourth, in cells lacking FRecQL5, we detected the presence of endogenous RecQL5 in the Pol II IP (Fig. (Fig.1d,1d, lane 6), although the level of RecQL5 was lower than that from cells overexpressing FRecQL5 (Fig. (Fig.1c,1c, compare lane 2 to lane 1). Our data are consistent with previous reports that RecQL5 associates with Pol II (3, 21).

FIG. 1.
A major component of a RecQL5 complex is Pol II. (a) Silver-stained SDS-PAGE gel of the RecQL5-Pol II complex isolated by Flag IP from HeLa cells expressing Flag-RecQL5 (FRecQL5). The three major polypeptides identified by mass spectroscopy analysis are ...

We analyzed the presence of other known RecQL5-interacting partners in our FRecQL5 IP by immunoblotting and observed the presence of Rad51, PCNA, and the MRN complex but not Topo3α and Topo3β (Fig. (Fig.1b,1b, lane 6, and see Fig. S2, lanes 8 and 10, in the supplemental material). Because these partners were not detected by mass spectrometry (unlike Pol II), they may associate with small fractions of RecQL5 compared to Pol II.

We also analyzed our Pol II IP for the presence of other RecQ helicases, such as WRN and BLM, but failed to detect any of them (Fig. (Fig.1d,1d, lane 6). This is consistent with early findings that the RecQL5-Pol II association is specific (3). Moreover, this association was not significantly altered by the treatment of cells with drugs that induce replication stress, such as camptothecin (CPT) and hydroxyurea (HU) (the ratio of Pol II over RecQL5 in the RecQL5 immunoprecipitate changes no more than 2-fold) (Fig. 1e and f).

RecQL5 interacts with Pol II through conserved KIX and SRI domains.

We mapped the Pol II-interacting region(s) in RecQL5 by deletion mutagenesis. Various Flag-tagged deletion mutants (Fig. (Fig.2a)2a) were transfected into HEK293 cells and analyzed by Flag IP and Western analyses. Two nonoverlapping regions outside the RecQL5 helicase domain were each found to be capable of associating with Pol II (Fig. 2b to d; summarized in Fig. Fig.2a).2a). The first region (residues 501 to 650) associated with both hypophosphorylated (IIa) and hyperphosphorylated (IIo) Pol II, whereas the second region (residues 745 to 991) interacted only with Pol IIo (Fig. (Fig.2d).2d). These data are consistent with a recent finding that RecQL5 has two independent Pol II-interacting regions (21).

FIG. 2.
RecQL5 interacts with Pol II through two conserved domains, the KIX and SRI domains. (a) Schematic diagram of the full-length (FL) and truncated forms of Flag-RecQL5. The N-terminal helicase and RecQ C-terminal (RQC) domains are shown along with the newly ...

Bioinformatic analyses revealed that each Pol II-interacting region contains a conserved protein interaction domain (11, 13, 17, 23, 32). The one in the first region (aa 540 to 620) is homologous to the KIX domain found in several Pol II transcriptional regulators (32), whereas the one in the second region (aa 909 to 991) is homologous to the SRI (Set2 Rpb1-interacting) domain present in the histone methyltransferase SetD2, which also regulates transcription (23) (Fig. (Fig.2a).2a). Interestingly, the SetD2-SRI domain is known to interact with the phosphorylated C-terminal domain (CTD) (pCTD) of Pol IIo, which is in complete agreement with the function of the predicted SRI domain in RecQL5.

Homology modeling predicts a strong structural similarity between RecQL5-KIX and other KIX domains.

Several KIX domain structures were previously determined, with all of them containing a KIX fold, a three-helix bundle with a conserved hydrophobic core (37, 40, 45, 48). Primary-sequence alignment revealed a high degree of similarity between RecQL5-KIX of different species and the KIX domains from several Pol II transcriptional regulators, CBP, p300, and Med15 (Fig. (Fig.3a).3a). Secondary-structure predictions identified three potential α-helices in RecQL5-KIX, consistent with the presence of the KIX fold (Fig. (Fig.3a3a and data not shown). Given the high degree of primary-sequence and secondary-structure similarity, a homology model for the RecQL5-KIX domain was generated by a standard protein comparison method using the mouse CBP (mCBP)-KIX domain (PDB accession number 1SB0) as a structural template (Fig. (Fig.3b)3b) (2, 48).

FIG. 3.
The RecQL5-KIX domain interacts with both Pol IIa and Pol IIo through a common interface utilized by other KIX domains. (a) Sequence alignment showing similarity between the RecQL5-KIX domain and KIX domain family members CBP, p300, and Med15. KIX family ...

Our model predicts that RecQL5-KIX contains a three-helix bundle similar to that of mCBP-KIX. Invariant hydrophobic positions within the core of the KIX fold are well conserved in primary sequence and in tertiary space between the two proteins (Fig. 3a and c). In contrast, surface-exposed hydrophobic residues are not conserved between RecQL5 and mCBP (Fig. 3a and c).

The KIX fold from all proteins contains two invariant charged residues and one invariant aromatic residue, all three of which are strictly conserved in RecQL5-KIX (R550, E584, and Y597) (Fig. 3a and d). The corresponding residues in mCBP (R600, E636, and Y649) form a stabilizing hydrogen bond network with several nearby hydrophobic core residues (V595 and Y640) (37) (Fig. (Fig.3d).3d). Interestingly, all residues of this extended hydrogen bond network are well conserved in RecQL5, suggesting that a similar network is present in RecQL5-KIX (Fig. (Fig.3d3d).

Mutations in the RecQL5-KIX domain disrupt its Pol II association.

A previous study showed that a mutation of the invariant glutamate residue within the hydrogen-bound network of Med15-KIX abolishes its interaction with its partner (SREBP-1a) (45). We found that mutations at comparable residues in RecQL5-KIX, E584D and E584A, also disrupted its interaction with the corresponding partners, Pol IIo and Pol IIa (Fig. (Fig.3g3g and data not shown). The data support our model that RecQL5-KIX contains a hydrogen bond network similar to that of other KIX domains and suggest that this network may be essential for all KIX domains to associate with their partners.

We next examined whether the E584 mutations can affect the association between full-length RecQL5 and Pol II. Because full-length RecQL5 consists of two independent Pol II interaction domains, the E584 mutations are expected to abrogate only the Pol IIa association (which is mediated solely by the KIX domain) but not the Pol IIo association (which can occur through either KIX or SRI). Consistent with this prediction, both E584D and E584A mutants lost their associations with Pol IIa but maintained their associations with Pol IIo (Fig. (Fig.3h,3h, lane 3, and data not shown). Moreover, mutations of another invariant residue within KIX, Y597A, reduced the Pol IIa association by about 70% while retaining the normal association with Pol IIo (Fig. (Fig.3h,3h, lane 7). Together, the data indicate that the KIX domain and the integrity of its hydrogen bond network are indispensable for the interaction between RecQL5 and Pol IIa but dispensable for the interaction with Pol IIo.

We found that a mutation of the third invariant residue of KIX, R550, strongly destabilized RecQL5 (Fig. (Fig.3h,3h, right, lane 6), suggesting that R550 is critical for the proper folding of the full-length protein. Interestingly, a previous study showed that a 9-residue deletion mutant, which includes R550, drastically decreased the association between RecQL5 and Pol II in vitro (4). Our RecQL5-KIX homology model suggests that this mutant lacked critical hydrophobic core residues and the first turn of helix 1, which could result in the destabilization of the native KIX domain.

RecQL5-KIX interacts with Pol II through a common protein interaction surface.

Next, we used the RecQL5-KIX domain homology model to predict the Pol II interaction surface. Unlike residues that maintain the integrity of the KIX fold, many of the surface-exposed residues that mediate direct intermolecular protein interactions are not conserved between RecQL5 and mCBP (Fig. 3a, e, and f). The largest contiguous RecQL5 conserved surface mapped along the interface of helix 1 and helix 3. Strikingly, mCBP-KIX and Med15-KIX use the same surface to bind their respective partners (Fig. 3a, e, and f) (10, 14, 25, 37, 45, 48).

To determine whether the predicted interface in RecQL5 is important for its interaction with Pol II, we independently mutated four residues on this surface, C553A, L556D, L602D, and K598A, in full length RecQL5 (Fig. 3a and e). IP-Western analysis showed that mutations of the first 3 residues reduced the Pol IIa association by more than 80% (Fig. (Fig.3h,3h, right, lanes 8, 9, and 11), supporting our model that RecQL5 employs the same protein-interacting surface as other KIX domains. As expected, all mutants retained the association with Pol IIo due to redundant interactions with the SRI domain.

The strongest reduction of the Pol II association (>95%) was observed with the RecQL5 L602 mutation, which corresponds to residues A654 in mCBP and A60 in Med15 (Fig. (Fig.3a).3a). Mutations of this residue in the latter KIX domains disrupt their interactions with their respective partners (37, 45, 48). In mCBP, A654 specifically recognizes a conserved leucine found in several CBP partners. Although RecQL5 has a leucine rather than an alanine at this position, the role of this residue in mediating protein interactions is likely conserved.

The interaction between the RecQL5-KIX domain and Pol II is specific.

Previous studies have shown that a KIX domain can interact with multiple partners and that several KIX domains can interact with the same partner (45). We examined whether other KIX domains can interact with Pol II by transfecting various Flag-tagged KIX domains into HEK293 cells, followed by IP-Western analysis. Only the KIX domain of RecQL5, but not those of CBP, p300, and Med15, associated with Pol II (Fig. (Fig.3i),3i), suggesting that the interaction between RecQL5-KIX and Pol II is specific. This specificity is consistent with our homology model showing that several residues at the protein-interacting surface of RecQL5-KIX are distinct from those in other KIX domains.

SRI domains of RecQL5 and SetD2 likely use a homologous interface to bind Pol IIo.

Primary-sequence alignment and secondary-structure prediction revealed strong similarity between the SRI domains of RecQL5 and SetD2 (Fig. (Fig.4a).4a). To further examine the relationship between the two domains, we generated a homology model of the RecQL5-SRI domain based on the structure of the human SetD2-SRI domain that was previously solved (PDB accession number 2A7O) (Fig. 4a and b) (2, 27). Our model predicts that RecQL5-SRI consists of the same structural fold, a three-helix bundle and a conserved hydrophobic core, as SetD2-SRI, although the length of the former is approximately 28 residues shorter than that of the latter. The regions that showed the strongest difference are the first two helices and the intervening loops, in which many residues conserved in SetD2-SRI are either different or absent in RecQL5-SRI (Fig. (Fig.4a).4a). Many of these residues are located along the periphery of the hydrophobic core or in structurally divergent sequences (Fig. 4a and c). Despite such differences, the helical segments comprising the main SRI fold are well conserved in RecQL5-SRI, including the essential hydrophobic core residues (Fig. 4a and c). Importantly, the surface-exposed residues, especially those critical for Pol II-pCTD binding (27), are invariant between the two SRI domains (Fig. 4a and d). We therefore hypothesize that RecQL5 binds Pol IIo using the same conserved surface as does SetD2-SRI (Fig. 4a and d).

FIG. 4.
The RecQL5-SRI domain interacts only with Pol IIo, and its structure is similar to that of the human SRI domain. (a) The sequence alignment shows strong similarity between the RecQL5 and SetD2 SRI domains. The color usage is described in the legend of ...

To test this hypothesis, we mutated two of the conserved surface residues (K939 and R943) of RecQL5-SRI to alanine. Mutations of the comparable residues in SetD2-SRI have been shown to strongly diminish its interactions with the Pol II-pCTD (27). IP-Western analysis showed that RecQL5-SRI domains carrying either K939 or R943 mutations failed to associate with Pol IIo (Fig. (Fig.4e),4e), supporting our hypothesis that RecQL5 binds Pol IIo-pCTD though the same interface utilized by SetD2.

Both the KIX and SRI domains in RecQL5 can independently bind Pol II.

We investigated the effect of SRI domain mutations on the association of full-length RecQL5 and Pol II. Two SRI domain deletion mutants (residues 1 to 900 and 1 to 650) retained normal associations with both forms of Pol II (Fig. (Fig.2b),2b), indicating that the SRI domain is dispensable for full-length RecQL5 to bind Pol II. This result is expected because both SRI deletion mutants retain the intact KIX domain that is capable of binding both Pol IIa and Pol IIo. Together with the finding that the KIX point mutant (E584D) retained its association with Pol IIo due to the presence of the SRI domain (Fig. (Fig.3h,3h, lane 3, and 4f, lane 3), our data suggest that both the KIX and SRI domains can independently bind Pol II.

One prediction from the above-described suggestion is that the inactivation of RecQL5-Pol II interaction requires simultaneous mutations of both the KIX and SRI domains. We generated two such mutants, E584D K939A and E584D R943A, and found that they substantially reduced associations with both forms of Pol II (Fig. (Fig.4f,4f, lanes 4 and 5). The results are in complete agreement with the notion that KIX and SRI domains bind Pol II independently and further indicate that there are no additional Pol II-interacting domains in RecQL5.

The RecQL5-Pol II association is conserved in chicken DT40 cells.

Next, we utilized chicken DT40 cells to investigate the significance of RecQL5-Pol II interactions in vivo. DT40 cells have a high gene-targeting efficiency and have been used widely for genetic analyses of DNA damage response factors (44), including RecQL5, BLM, RecQL1 (31), and multiple Fanconi anemia proteins. The findings from these analyses were invaluable and they led directly to the discovery of a new Fanconi anemia gene (26). One reason for this is that human and chicken proteins are highly homologous in sequence, so their functions are well conserved.

For RecQL5, both its KIX and SRI domains are highly conserved between human and chicken, with about 60% identity and over 70% similarity (Fig. (Fig.3a3a and and4a).4a). More importantly, the residues predicted to interact with Pol II are strongly conserved, with over 70% identity and 93% similarity for the KIX domain (Fig. (Fig.5a)5a) and 100% identity for the SRI domain (Fig. (Fig.5b).5b). For Pol II, it is known to be one of the most highly conserved proteins in all eukaryotes. For example, the RBP1 subunit of Pol II is over 90% identical and 95% similar between human and zebrafish. Because both Pol II and its interaction domains in RecQL5 are highly conserved between human and chicken, we predict that their association is also conserved in DT40 cells.

FIG. 5.
The association between RecQL5 and Pol II is conserved in chicken DT40 cells. (a and b) RecQL5-KIX (a) and RecQL5-SRI (b) human and chicken conserved Pol II interaction surfaces. (a) Ribbon model of human RecQL5-KIX shown as described in the legend of ...

Consistent with this prediction, human RecQL5 transfected into DT40 cells coimmunoprecipitated with both Pol IIa and IIo (Fig. (Fig.5c).5c). Moreover, various KIX and SRI mutants of RecQL5 displayed the same Pol II interaction patterns in DT40 cells as those in human HEK293 cells (compare Fig. Fig.5c5c with with3h3h and 4e and f). For example, the KIX domain point mutant (E584D) in DT40 cells similarly exhibited a reduced association with Pol IIa but retained normal associations with Pol IIo. The SRI domain deletion mutant (ΔSRI) had associations with both Pol IIa and Pol IIo. The double point mutants of the KIX and SRI domains (E584D K939A) lost associations with both Pol II forms. These data suggest that the mechanism of RecQL5-Pol II interactions is conserved in chicken, which allows the use of DT40 cells for functional analyses of these interactions.

We found that human RecQL5 transfected into DT40 cells coimmunoprecipitated with several other interacting partners of RecQL5, including RAD51 and PCNA (Fig. (Fig.5d,5d, lane 5). The data suggest that the interactions between RecQL5 and its other partners are also conserved in chicken.

The KIX but not the SRI domain is required for the suppression of SCE by RecQL5.

A previous study showed that RecQL5//BLM/ DT40 cells had an SCE level that was higher than that of BLM/ cells (41). We repeated these experiments and obtained similar results (Fig. (Fig.6b6b and see Fig. S3 in the supplemental material). The introduction of human RecQL5 into RecQL5//BLM/ cells restored the SCE level to that of BLM/ cells (Fig. 6a and b), indicating that RecQL5 is responsible for the elevated levels of SCE in these cells. This allowed us to test various RecQL5 mutants using the same assay to determine the importance of the different activities of RecQL5 in cells. Control experiments showed that all mutants were expressed at levels similar to or above that of the wild-type protein (Fig. 5c and d).

FIG. 6.
Both helicase activity and KIX domain-mediated Pol IIa interactions are required for RecQL5 to suppress SCE and resist CPT-induced cell killing. (a) Summary of different human RecQL5 mutants and their corresponding activities in association with Pol II, ...

We found that the KIX domain E584D mutant partially suppressed the abnormal SCE phenotype of RecQL5//BLM/ cells (Fig. 6a and b). In contrast, the SRI domain deletion mutant (ΔSRI) fully suppressed the abnormal SCE phenotype (Fig. (Fig.6b).6b). Moreover, the KIX-SRI double mutant (E584D K939A) suppressed SCE to a level similar to that by the KIX domain single mutant (E584D) (Fig. (Fig.6b).6b). These data demonstrate that the two Pol II interaction domains in RecQL5 are functionally nonequivalent in the suppression of SCE: the KIX domain is essential, whereas the SRI domain is dispensable.

The KIX but not the SRI domain is required for cellular resistance to camptothecin.

Mouse RecQL5/ cells have been shown to exhibit hypersensitivity to CPT, a topoisomerase I inhibitor that blocks replication (19). However, neither RecQL5/ nor BLM/ DT40 cells showed significant CPT sensitivity, whereas RecQL5//BLM/ double-mutant cells displayed the sensitivity (Fig. (Fig.6c).6c). The feature is reminiscent of the SCE data in that only the double mutant, but not the RecQL5 single mutant, displayed a defective phenotype. The data suggest that RecQL5 and BLM have redundant functions not only in the suppression of crossover recombination but also in the resistance of replication stress.

We tested RecQL5 and its mutants using the CPT sensitivity assay. The transfection of human RecQL5 into RecQL5//BLM/ cells largely restored cellular resistance to CPT (Fig. 6a and d). In addition, the KIX domain mutant (E584D) partially restored CPT resistance, whereas the SRI domain mutant (ΔSRI) almost fully corrected CPT resistance (Fig. 6a and d). Furthermore, the double mutant of both domains, E584D K939A, had an effect similar to that of the KIX single mutant. These data are in complete agreement with findings from the SCE assay and suggest that the KIX domain is required for not only SCE suppression but also CPT resistance, whereas the SRI domain is dispensable for both.

The RecQL5 helicase and KIX domains are both required for full suppression of SCE and resistance to CPT.

RecQL5 possesses a highly conserved helicase domain and an associated helicase activity, but their importance in vivo remains unclear. To address this issue, we generated two helicase mutants and investigated if they can complement the abnormal SCE and CPT sensitivity phenotypes of RecQL5//BLM/ cells. The first mutant (K58R) was shown previously to lack ATP-dependent helicase activity (15), whereas the second mutant (ΔHel) had a majority of its helicase domain deleted (residues 1 to 241) so that it should lack both ATP-dependent helicase activity and ATP-independent DNA-binding activity. Both mutants retained normal associations with Pol IIa and IIo (Fig. (Fig.5c,5c, lanes 5 and 7, and d, lane 7), which is expected given the presence of the intact KIX and SRI domains. Notably, both mutants only partially restored the abnormal SCE level and CPT resistance of RecQL5//BLM/ DT40 cells compared to the wild-type protein (Fig. 6a, b, and e), suggesting that the helicase activity is important for RecQL5 to function in vivo. The fact that these helicase mutants had partial activity in correcting the SCE and CPT phenotypes suggests that RecQL5 can act through a mechanism distinct from that of its helicase activity.

We noticed that the Pol IIa interaction mutant (E584D) also had partial activity in restoring SCE levels and CPT resistance of RecQL5//BLM/ DT40 cells, a feature reminiscent of that of the helicase mutants. This implies that the helicase activity and Pol IIa interaction are parallel in protecting genome stability. To test this hypothesis, we created a double mutant, K58R E584D, to inactivate both its helicase activity and Pol IIa interaction. RecQL5//BLM/ cells complemented by this mutant had SCE levels and CPT sensitivity indistinguishable from those of double-null cells (Fig. 6a, b, and e). The data suggest that RecQL5 can promote genome stabilization through either its helicase activity or Pol IIa association. Only when both activities are disrupted does RecQL5 completely lose its function.

As a control, the E584D, K58R, and K58R E584D mutants associated with normal levels of RAD51, PCNA, and the MRN complex by IP-Western analyses of HEK293 cell extracts (see Fig. S2, lanes 9 to 12, in the supplemental material). Moreover, the same mutants also associated with normal levels of RAD51 and PCNA in DT40 cell extracts (Fig. (Fig.5d,5d, lanes 5 to 8). These data argue that a loss of function of these mutants is not due to the disruption of their association with other partners of RecQL5.

DISCUSSION

This study addresses three main issues regarding how RecQL5 functions in genome stabilization. First, which interacting partners of RecQL5 are functionally relevant? Second, what structural domains of RecQL5 are responsible for interacting with its partner(s)? Third, is the helicase activity of RecQL5 required? To answer these questions, we independently purified a RecQL5 complex and identified Pol II as its main component. Using DT40 cells inactivated for RecQL5, we demonstrated that Pol II is a critical partner of RecQL5 in the suppression of SCE and resistance of CPT-induced cell death. Moreover, we identified two conserved domains in RecQL5, KIX and SRI, and showed that they are responsible for interacting with Pol II. Furthermore, we found that the helicase activity is required by RecQL5 to function normally in vivo. Finally, we presented evidence that the helicase action and Pol II interaction are two independent mechanisms of RecQL5 in promoting genome stabilization.

RecQL5 requires the KIX but not the SRI domain for maintaining genome stability.

We found that the two Pol II-interacting domains of RecQL5 are functionally nonequivalent. Only the KIX domain is required for RecQL5 to suppress SCE and resist CPT-induced cell killing. In contrast, the SRI domain is completely dispensable, as its point mutation or deletion has no significant effects on either function of RecQL5 in vivo (Fig. (Fig.6).6). There are two possible explanations for the observed difference. One possible explanation is that the difference is due to the different binding capacities of the two domains for Pol II. The KIX domain can bind both Pol IIa and Pol IIo, whereas the SRI domain can interact only with Pol IIo. As a result, RecQL5 without the SRI domain can still associate with both forms of Pol II (Fig. (Fig.2b),2b), whereas RecQL5 with a mutated KIX domain fails to associate with Pol IIa. This allows the KIX domain to substitute functionally for a defective SRI domain but not vice versa. An alternative explanation is that only the KIX domain-mediated interaction with Pol II can regulate the polymerase activity, whether Pol IIa or Pol IIo, whereas the SRI-mediated interaction is nonfunctional. Future studies will be necessary to distinguish these possibilities and determine whether the SRI domain plays a role in other cell types and species.

RecQL5 may protect genome integrity by inhibiting transcription initiation.

RecQL5 was shown to be capable of inhibiting Pol II transcription at both initiation and elongation steps in vitro (4). In agreement with this, we found that RecQL5 can bind both the initiation (Pol IIa) and elongation (Pol IIo) forms of the polymerase (34) through the KIX and SRI domains. However, which of the two steps requires regulation by RecQL5 to protect genome integrity in vivo remains unknown. Our analyses of various RecQL5 mutants using SCE and CPT sensitivity assays favor a hypothesis that the step that requires RecQL5 regulation could be initiation. For example, the KIX domain mutant (RecQL5-E584D), which lacks the association with Pol IIa but retains associations with Pol IIo, is deficient in SCE suppression and CPT resistance, suggesting that the interaction with the initiation polymerase (Pol IIa) is important for RecQL5 to function in vivo. In contrast, the deletion or point mutation of the SRI domain, which specifically binds the phosphorylated CTD of the elongation polymerase (Pol IIo), has no obvious effects on the SCE- or CPT-associated phenotypes, arguing that the RecQL5 interaction with the elongation polymerase is dispensable.

We emphasize that our data do not exclude an alternative possibility, that RecQL5 also inhibits transcription elongation in vivo. This may occur through its KIX domain, which can interact with both Pol IIa and Pol IIo, resulting in the inhibition of Pol II activity during both initiation and elongation. However, the inhibition of transcription at the initiation stage has several advantages. First, it will prevent the opening and modification of chromatin that accompany Pol II elongation. The opening of the chromatin structure may enhance the mutation of the genome by making DNA more accessible to damaging agents such as nucleases. It may also increase recombination events by exposing recombination-prone repeat sequences (1). Second, the inhibition of initiation may decrease the number of transcription bubbles and reduce their head-on collisions with replication forks, which could impair the progression of forks and lead to increased transcription-associated recombination (36). Third, the inhibition of initiation may also decrease the arrays of transcription complexes backed up by a lesion on the template DNA, thus allowing a replication fork to directly access and eventually bypass the lesion (38).

RecQL5 promotes genome stabilization through two parallel mechanisms: acting as a helicase and interacting with Pol II.

Our structural and functional analyses of RecQL5 suggest that it can protect genome integrity by two independent mechanisms: acting as a RecQ helicase through its helicase domain and regulating Pol II activity through its KIX domain. In support of this model, the mutation of either domain results in a partial deficiency in the suppression of SCE and CPT resistance, whereas the simultaneous inactivation of both domains leads to a complete deficiency. The latter result also implies that other interacting partners of RecQL5 may not play significant roles in RecQL5 function, at least in DT40 cells. The notion that RecQL5 functions through two independent mechanisms is consistent with a previous finding that the helicase domain of RecQL5 is dispensable for the inhibition of Pol II-dependent transcription in vitro (4). It is also in agreement with data showing that a 9-residue KIX domain deletion mutant, which has intact helicase activity but is defective in Pol II interaction, fails to inhibit transcription in vitro.

How may RecQL5 promote genome stabilization through two mechanisms? One can hypothesize several possibilities susceptible to direct experimental tests. In the suppression of SCE, RecQL5 may use its intrinsic helicase activity to directly decrease HR events by dissociating D loops and RAD51 filaments. Alternatively, RecQL5 may indirectly downregulate HR by inhibiting transcriptional initiation. It was shown previously that transcription can stimulate intrachromosomal HR and VDJ recombination (8, 24, 30). The downregulation of HR should ultimately lead to reduced levels of SCE. In stabilizing and restarting blocked forks, RecQL5 may use its helicase activity to prevent blocked forks from collapse by catalyzing fork reversal. The resulting Holliday junction can be subsequently repaired by a template-switching mechanism (9). On the other hand, RecQL5 may inhibit Pol II activity to reduce head-on collisions between transcription and replication machinery, leading to fewer stalled forks (36, 38). The ability of RecQL5 to regulate recombination, replication, and transcription makes it a crucial guardian of genome integrity.

Supplementary Material

[Supplemental material]

Acknowledgments

We thank A. Naar for providing CBP-KIX and Med15-KIX vectors, P. Wright for providing mouse CBP-KIX, and M. Gorospe for providing the p300 vector. We also thank D. Schlessinger for critical reading of the manuscript and the National Cell Culture Center for providing cells.

This work was supported in part by the Intramural Research Program of the National Institute on Aging (Z01:AG000657-09), National Institutes of Health. The work of the group of W.W. has also been supported by a grant from the Fanconi Anemia Research Fund.

Footnotes

[down-pointing small open triangle]Published ahead of print on 15 March 2010.

Supplemental material for this article may be found at http://mcb.asm.org/.

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