RecQ5 Protein Translocation into the Nucleus by a Nuclear Localization Signal

Abstract

RecQ5, a member of the RecQ helicase family, maintains genome stability via participation in many DNA metabolic processes including DNA repair, DNA resolution, and RNA transcription, processes occurring in the nucleus. Previously, we reported that RecQ5 and Rad51, also involved in DNA repair, become co-localized in nuclei when co-expressed in cultured cells. Nuclear localization of RecQ5 appears to be important for cellular function along with Rad51. However, little is known about the nuclear localization of RecQ5. Here, we generated enhanced green fluorescent protein (EGFP)-tagged RecQ5 transgenic flies and analyzed localization of this protein in early embryos by live imaging. In syncytial embryos, RecQ5 was localized synchronously in interphase nuclei, and spread repeatedly over the embryos in mitosis. Thus, RecQ5 was transported into nuclei at the early interphase. Furthermore, we examined the subcellular localization of a series of truncated forms of Drosophila RecQ5 in cultured cells to determine the nuclear localization signal (NLS). Entire coding or deleted RecQ5 sequences of various sizes were ligated into EGFP vectors, which were then used to transfect cultured Drosophila cells. The region responsible for nuclear localization of Drosophila RecQ5 contained a short stretch of positively charged basic amino acids, 2 of which were particularly important for the nuclear localization. This stretch was sufficient for nuclear localization when fused with EGFP. Although the NLS of Drosophila RecQ5 was distinct from that of human RECQL5 in terms of position and amino acid sequence, this fly RecQ5 protein was translocated into the nucleus by an NLS.

RecQ5 DNA helicase is a member of the RecQ family, in which there are 5 members in humans. Three of them are involved in predisposition to cancer, premature aging and/or developmental abnormality diseases such as Bloom and Werner syndromes, which are caused by mutations in the BLM and WRN genes, respectively; and 3 other syndromes, i.e., Rothmund–Thomson, RAPADALINO, and Baller–Gerold syndromes, are caused by mutations in the RecQ4 gene.^1–4) Although RecQL1 and RECQL5 (human homolog of Drosophila RecQ5), the other 2 members of the RecQ family in humans, have not been associated with any human genetic diseases, Recql5-knock out mice show susceptibility to cancer,⁵⁾ suggesting that RecQ5 may also play an important role in preventing cancer. Furthermore, it is postulated that RECQL5 has multiple activities in transcription with RNA pol II,^6,7) homologous recombination with MRN and Rad51,^8,9) and DNA decatenation with Topo II.¹⁰⁾ These activities are elicited in the nucleus. Nuclear localization of RecQ5 may be important for these activities. However, the precise nuclear transport mechanism of RecQ5 remains unknown.

In Drosophila, the frequencies of spontaneous and induced chromosomal aberrations are increased in RecQ5-mutant neuroblasts.¹¹⁾ These data imply that double-stranded break (DSB) damage to DNA accumulates spontaneously in RecQ5 mutants. Furthermore, the loss of maternal RecQ5 leads to spontaneous mitotic defects in syncytial embryos.¹²⁾ These mitotic defects are derived from anaphase DNA bridges, which link pairs of daughter nuclei. These nuclei concurrently exit from the cycle and are eliminated by Drosophila checkpoint kinase 2 tumor suppressor homolog (DmChk2)-dependent centrosome inactivation. DmChk2 responds to DSB DNA lesions.¹²⁾ These results suggest that the lack of RecQ5 leads to spontaneous DSBs. RecQ5 may function in the resolution of anaphase DNA bridges during mitosis or in DSB repair during the interphase in syncytial Drosophila embryos. However, it remained unknown whether RecQ5 protein localizes in anaphase DNA bridges, mitotic chromosomes or interphase nuclei in syncytial embryos.

Previously, we showed that DNA-damaging agents, such as methyl-methane sulfonate and cisplatin, increase the level of RecQ5 protein in S2 cells¹³⁾ and that recq5 mutant flies are sensitive to cisplatin.¹⁴⁾ In addition, Rad51 protein interacts with RecQ5 in vitro and in vivo.¹⁴⁾ Rad51 itself, which plays a critical role in DSB repair, has no detectable nuclear localization signal (NLS). When RecQ5 and Rad51 are co-expressed in Drosophila cells in culture, they become co-localized in the nuclei.¹⁴⁾ Nuclear import of the Rad51 protein likely requires its interaction with another protein that contains a functional NLS, such as RecQ5. The nuclear import of RecQ5 appears to be important for RecQ5 function as well as for the function of other proteins, including Rad51. However, nuclear import of RecQ5 had not been previously elucidated in detail. In fact, no NLS had been identified in Drosophila RecQ5.

Here, we observed the behavior of RecQ5 in Drosophila syncytial embryos by live imaging, and examined its subcellular localization by preparing a series of truncated forms of Drosophila RecQ5 in cultured cells. The region identified to be responsible for the nuclear localization of DmRecQ5 contained a short stretch of positively charged basic amino acids. Two of these basic amino acids were especially important for the nuclear localization. This stretch was sufficient for the nuclear localization when it was fused with the enhanced green fluorescent protein (EGFP). The NLS of Drosophila RecQ5 was distinct from that of human RECQL5 in terms of position and amino acid sequence.

Materials and Methods

Cell Culture and Transfection

Cells of the Drosophila embryo-derived cell line D.Mel-2 (Life Technologies) were cultured in Express Five SFM medium (Life Technologies) supplemented with 9% of 100×GlutaMAX (Life Technologies) under the 28°C condition.

Transfection and Fluorescence Microscopy Analysis

Cells grown on Lab-Tek II Chamber Slides (NUNC) were transfected with pWA-Gal4 (a kind gift from Y. Hiromi) as the effector plasmid and pUAST¹⁵⁾ constructs by using Cellfectin II reagent (Life Technologies). Forty-eight hours after transfection, the cells were fixed with 4% formaldehyde in phosphate-buffered saline (PBS) for 15 min, and permeabilized with 0.2% Triton X-100 in PBS for 5 min. After having been washed with PBS, the samples were blocked with 2% bovine serum albumin (BSA) in PBS for 30 min. The samples were then washed again with PBS, air-dried, and mounted in ProlongGold with 4′,6-diamidino-2-phenylindole (DAPI) (Life Technologies). The cells were observed under a confocal laser-scanning microscope (FV-1000, OLYMPUS). FLAG-tag was detected as previouly described.¹⁴⁾

Plasmid Construction

To construct a series of plasmids expressing EGFP-RecQ5, we fused various deletion RecQ5 cDNA fragments with the EGFP cDNA sequence, amplified them by polymerase chain reaction (PCR), and inserted them into pUAST. RecQ5^1–408-FLAG was generated by the fusion of RecQ5 cDNA Eco52I fragment and FLAG-tag in pUAST. EGFP-RecQ5^R501A, EGFP-RecQ5^K503A, EGFP-RecQ5^K504A, EGFP-RecQ5^{R501A K503A}, EGFP-RecQ5^{K500A R501A K503A}, and EGFP-RecQ5^{R501A K503A K504A} were generated by using a QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies).

Generation of EGFP-Tagged RecQ5-Expressing Transgenic Flies

The 5′ portion of the RecQ5 gene was cloned by using primers 5′-TGC CTG CAG GGT TTT TGG GGA ATT TCG AT-3′ and 5′-TAC TGC AGT TGC TTC AGA CCA AAA CTG G-3′, to amplify a DNA fragment from Drosophila genomic DNA by PCR. DNA fragments containing the 5′-portion of the RecQ5 gene were inserted into the PstI/EcoRI site of pUAST to generate pQ5^P. pQ5^P-EGFP-RecQ5 was constructed by the insertion of EGFP-RecQ5 into pQ5^P. Four independent transgenic lines were generated (q5^P-egfp-recq5).

Results

Localization Analysis of EGFP-RecQ5 in Early Embryos by Real-Time Imaging

To visualize the subcellular localization of RecQ5, we generated transgenic flies expressing EGFP-tagged RecQ5 under the control of its own promoter, and analyzed the expression pattern of RecQ5 in embryos. EGFP from the jellyfish Aequorea victoria yields a strong fluorescent signal in heterologous cell types and has been used for gene expression and visualizing protein translocation into subcellular organelles in living cells. The EGFP-RecQ5 was expressed in early embryos (q5^P-egfp-recq5), consistent with the results showing RecQ5 expression in early embryos.¹⁶⁾ We generated q5^P-egfp-recq5;His2AvD-mRFP1 flies to visualize both RecQ5 and chromatin in living embryos.¹²⁾ The mRFP1 (monomeric red fluorescent protein from Discosoma) shows adequate brightness in living cells. In addition, the excitation and emission peaks of mRFP1 confer greater tissue penetration and spectral separation from autofluorescence and other fluorescent proteins.¹⁷⁾ Drosophila His2AvD, a variant of histone protein H2A, is required for the survival of Drosophila melanogaster. A His2AvD-GFP fusion gene complements a lethal His2AvD mutant allele and provides an in vivo marker of Drosophila chromosome behavior.¹⁸⁾ In interphase cells, the EGFP-RecQ5 was colocalized with mRFP-tagged His2AvD (Fig. 1A), suggesting that RecQ5 had been localized in the nuclei. A mitotic wave began from both ends of the embryo. The EGFP-RecQ5 became dispersed in the embryo with the progression of the mitotic wave (Fig. 1B) and was excluded from metaphase chromosomes (Fig. 1C). Then from both ends of the embryo, the EGFP-RecQ5 synchronously entered into the nuclei (Figs. 1D, E). This process was repeated several times (supplemental movie). Thus, RecQ5 was localized only in S-phase nuclei, but not associated with mitotic chromosomes during stage 9 to 14 (Fig. 1, supplemental movie). Thus RecQ5 was transported into and became localized in nuclei during interphase. These data suggest that the localization of RecQ5 depended on the nuclear membrane formation and that RecQ5 functioned in the interphase nuclei.

Fig. 1. Localization Analysis of EGFP-RecQ5 in Early Embryos by Real-Time Imaging

Live embryos expressing EGFP-RecQ5 and His2AvD-mRFP1, which afforded visualization of RecQ5 (green) and histone (red), respectively, were analyzed by time-lapse confocal microscopy as described previously.¹²⁾ Images were obtained for 30–90 min after egg laying with a supply of maternally-derived EGFP-RecQ5 and mRFP-His2AvD. In syncytial embryos, no zygotic expression occurs; and maternal proteins drive the syncytial embryonic process. The cell-cycle length is about 10 min. Each panel represents interphase at cycle 10 (A), interphase to mitosis (B), mitosis (C), mitosis to interphase (D), and interphase at cycle 11 (E). Scale bar, 100 µm.

Putative NLS in the C-Terminus of RecQ5

Although some domains of RecQ5, such as the helicase domain, RQC/HCR-1 (the extended region homologous in the RecQ family/highly conserved region 1), KIX/HCR-2 (CREB binding homologous domain/highly conserved region 2), and SPI/HCR3 (Set2-Rpb1-interacting domain/highly conserved region 3) are conserved between humans and Drosophila,^19,20) other parts of the C-terminal region are not conserved (Fig. 2A, white boxes). A fragment of the C-terminus (aa 746–aa 991) of human RECQL5 migrated into the nucleus, indicating that the NLS of the HsRECQL5 protein resided within this C-terminal fragment.²¹⁾ A potential bipartite type of NLS with the sequence KRPRSQQENPESQPQKRPR exists in the C-terminus of human RECQL5²¹⁾; and also a monopartite NLS was predicted by NLS prediction programs (PSORT http://psort.hgc.jp/, PredictNLS http://www.predictprotein.org/, cNLS Mapper http://nls-mapper.iab.keio.ac.jp/, Fig. 2B). However, amino acid sequences of Drosophila RecQ5 in the region from aa 786 to aa 1058, corresponding to the C-terminal fragment of HsRECQL5, showed only a little conservation (Fig. 2B, Clustal omega http://www.clustal.org/omega/); and no NLS candidate was pointed out by the NLS prediction programs in this region of Drosophila RecQ5. Therefore, we sought to experimentally determine the existence of an NLS in Drosophila RecQ5.

Fig. 2. Subcellular Localization of DmRecQ5 in Cultured Cells

(A) Schematic diagram of HsRECQL5 and DmRecQ5 wild-type, and N-terminal deletion derivatives of the latter used to determine the subcellular localization of DmRecQ5. The closed, horizontal-striped, and hatched boxes indicate the helicase domain, the extended region homologous in the RecQ family (RQC/HCR-1), and the region homologous in the RecQ5 family (KIX/HCR-2 and SPI/HCR3), respectively.^19,20) EGFP was fused with the N-terminal of the derivatives. Numbers indicate sequence position of amino acids. (B) Sequence comparison of NLS candidate region of HsRECQL5 with DmRecQ5. Amino acid sequences of HsRECQL5, aa 854–aa 874, are shown with NLS candidates predicted by PSORT (underline), PredictNLS (bold), and cNLS Mapper (italic). DmRecQ5 region at aa 933 to aa 951, corresponding to the same region of HsRECQL5, was aligned by using Clustal omega. These amino acid residues are coloured as according to their physicochemical properties: Black, small amino acid residue; blue, acidic amino acid residue; red, basic amino acid residue; green, hydroxyl, sulfhydryl or amine amino acid residue. (C) Subcellular localization of EGFP-RecQ5 in D.Mel-2 cells. Confocal laser microscopy of D.Mel-2 cells transfected with the following constructs: EGFP-RecQ5^1–1058 (A–C), EGFP-RecQ5^109–1058 (D–F), EGFP-RecQ5^308–1058 (G–I), EGFP-RecQ5^421–1058 (J–L), EGFP-RecQ5^655–1058 (M–O), and EGFP-RecQ5^771–1058 (P–R) as described in Materials and Methods. DNA was stained with DAPI (blue). Scale bar, 10 µm.

Subcellular Localization of Full-Length RecQ5 and Its Deletion Mutants in D.Mel-2 Cells

To search for an NLS in Drosophila RecQ5, we constructed a set of truncated forms of RecQ5 mutants (Fig. 2A, deletion from N-terminal). D.Mel-2 cells were transfected with the EGFP vector harboring the entire coding sequence of RecQ5, which express RecQ5 well; and EGFP-RecQ5 (aa 1–aa 1058) was localized in the nucleus (Fig. 2C). The truncated forms of RecQ5 containing the region aa 109–aa 1058, aa 308–aa 1058, and aa 421–aa 1058 also became localized in the nuclei (Fig. 2C). In contrast, the RecQ5 mutants containing aa 655–aa 1058 and aa 771–aa 1058 remained localized in the cytoplasm (Fig. 2C). These results suggest that Drosophila RecQ5 contained an NLS located between aa 421 and aa 654.

In the case of the C-terminally deleted RecQ5s (Fig. 3A), truncated EGFP-tagged RecQ5s (aa 1–aa 963, aa 1–aa 749, and aa 1–aa 618) were localized in the nuclei (Fig. 3B). However, FLAG-tagged RecQ5 (aa 1–aa 468) remained in the cytoplasm, while FLAG-tagged whole RecQ5 (aa 1–aa 1058) is localized in the nuclei as reported previously.²²⁾ These data indicate that a region between aa 469 and aa 618 functioned as the NLS in Drosophila cultured cells.

Fig. 3. Examination of the Role of the C-Terminal Region of DmRecQ5 in the Nuclear Localization

(A) Schematic diagram of DmRecQ5 C-terminal deletion derivatives used to determine the subcellular localization of DmRecQ5. (B) Confocal laser microscopy of D.Mel-2 cells transfected with the following constructs: EGFP-RecQ5^1–963 (A–C), EGFP-RecQ5^1–749 (D–F), EGFP-RecQ5^1–618 (G–I), and RecQ5^1–468-FLAG (J–L). DNA was stained with DAPI (blue) as described in Materials and Methods. Scale bar, 10 µm.

To determine more precisely the region of DmRecQ5 required for the nuclear localization, we constructed an additional set of truncated forms of RecQ5 mutants (Fig. 4A). Nuclear localization of aa 421–aa 557 was observed as the smallest RecQ5 fragment (Fig. 4B). In contrast, the truncated fragments at 538 amino acid (aa 538–aa 557, aa 538–aa 618) remained in the cytoplasm (Fig. 4B).

Fig. 4. Dissection of Central Region of DmRecQ5 Required for the Nuclear Localization

(A) Schematic diagram of DmRecQ5 mutants used to determine the nuclear localization of DmRecQ5. (B) Confocal laser microscopy of D.Mel-2 cells transfected with the following constructs: EGFP-RecQ5^421–557 (A–C), EGFP-RecQ5^538–618 (D–F), and EGFP-RecQ5^538–557 (G–I). DNA was stained with DAPI (blue) as described in Materials and Methods. Scale bar, 10 µm.

Combining the above data, therefore, the minimum fragment for nuclear localization was between aa 469 and aa 537.

Identification of NLS in RecQ5

The region (aa 469–aa 537) identified as responsible for nuclear localization of RecQ5 contained a short stretch of positively charged basic amino acids (K, R, Fig. 5A). Although no potential NLS motif was predicted in the region aa 469–aa 537 by PSORT, we found a short stretch of positively charged basic amino acids (aa 500–aa 504, Fig. 5A). We substituted the basic amino acid to alanine in this region (Fig. 5B). Single amino acid substitutions R501A, K503A, and K504A, did not change its nuclear localization (Fig. 5C). However, double substitutions R501A and K503A caused the helicase to remain in the cytoplasm (Fig. 6C). In addition, triple substitutions K500A R501A K503A and R501A K503A K504A also kept RecQ5 in the cytoplasm (Fig. 5C). Therefore, R501 and K503 were important for nuclear localization of RecQ5.

Fig. 5. Amino Acid Substitution in RecQ5 NLS Sequence

(A) Amino acid sequence responsible for nuclear localization of RecQ5 (aa 469–aa 537) . The region contains a short stretch of positively charged basic amino acids (K and R, red). Underline indicates aa 500–aa 504 region. (B) Schematic diagram of amino acid substitution mutants used to determine the nuclear localization of DmRecQ5. R501A, K503A, K504A, R501A K503A, K500A R501A K503A, and R501A K503A K504A are indicated. Positively charged basic amino acids are marked in red. (C) Confocal laser microscopy of D.Mel-2 cells transfected with the following constructs: EGFP-RecQ5^R501A (A–C), EGFP-RecQ5^K503A (D–F), EGFP-RecQ5^K504A (G–I), EGFP-RecQ5^R501AK503A (J–L), EGFP-RecQ5^{K500AR501AK503A} (M–O), and EGFP-RecQ5^{R501AK503AK504A} (P–R). DNA was stained with DAPI (blue) as described in Materials and Methods. Scale bar, 10 µm.

In order to examine the role of this potential NLS motif within amino acids 469–537 of RecQ5, we introduced plasmids in which this putative NLS was fused with EGFP into D.Mel-2 cells (Fig. 6A). The EGFP fused with SHSSMAKRAKKESQDFI (aa 495–aa 509) became localized in the nuclei (Fig. 6B). In contrast, the expressed EGFP alone was localized also in cytoplasm (Fig. 6B). The short fragments might pass through the nuclear pores freely and become distributed in both the cytoplasm and the nucleus as does the EGFP, because the fragment has a low molecular weight (26.9 kDa, Fig. 6B). The stretch SHSSMAKRAKKESQDFI (aa 495–aa 509) was thus sufficient for the nuclear localization when it was fused with EGFP (Fig. 6B) and thus may be considered to be the NLS in RecQ5.

Fig. 6. Effect of RecQ5 NLS Sequence

(A) Schematic diagram of EGFP and its fusion protein with the 15-amino acid sequence fragment (EGFP-HSSMAKRAKKESQDF) used to determine the nuclear localization of DmRecQ5. (B) Confocal laser microscopy of D.Mel-2 cells transfected with only EGFP (A–C) or EGFP–HSSMAKRAKKESQDF (D–F). DNA was stained with DAPI (blue) as described in Materials and Methods. Scale bar, 10 µm.

Therefore, the RecQ5 protein has a functional NLS and is transported into the nucleus. The NLS of Drosophila RecQ5 was distinct from that of human RECQL5 in terms of both position and amino acid sequence.

Discussion

In this study, RecQ5 was localized only in interphase nuclei, and spread over the embryo in mitosis, repeatedly in living syncytial embryos (Fig. 1). Our study revealed a rapid nuclear transport of RecQ5 in living syncytial embryos. Our fluorescence analysis using cultured cells confirmed the finding of others^21,22) that RecQ5 is found in the nucleus in transfected cell lines. By studies on deletion mutants starting from the N-terminus (Fig. 2), NLS was located between aa 421–aa 654, and not in the region of aa 655–aa 1058 of RecQ5. Based on studies with deletion mutants made from the C-terminus (Fig. 3), the NLS was restricted to the region covering aa 469–aa 618, but not in aa 1–aa 468, of RecQ5. By using the central RecQ5 fragments (Fig. 4), the NLS was narrowed down to the sequence of aa 421–aa 537, not to that of aa 538–aa 618, of RecQ5. From the results found for these truncated RecQ5 molecules, the NLS may have resided in sequence aa 469–aa 537, but not in aa 1–aa 468, aa 538–618 or aa 655–aa 1058. There remained a possibility that another NLS existed in aa 619–aa 654, though no candidate was predicted (cNLS Mapper, PSORT and PredictNLS). Furthermore, the substitution including 2 positively charged basic amino acids (R501, K503) abolished the nuclear localization of RecQ5 (Fig. 5), suggesting that both of these residues were essential for a functional NLS. In addition, the sequence of 15 amino acids (aa 495–aa 509) was sufficient to localize EGFP in the nucleus (Fig. 6). Therefore, this sequence would be the NLS in RecQ5.

As previously stated, the NLS of Drosophila RecQ5 was distinct from that of human RECQL5 in both position and amino acid sequence. However, the NLS, aa 495–aa 509, of Drosophila melanogaster RecQ5 was found to be well conserved in 12 Drosophila species²³⁾ (Fig. 7); whereas amino acids in other parts of the C-terminal region of RecQ5 were varied even in 12 related species. Especially, the core region around KRAKK (aa 500–aa 504) region was almost identical in them (SH—AKRAKKE—FI), suggesting that this core region was important for the nuclear localization of RecQ5. The NLS of Drosophila RecQ5 contributes to the accumulated experimental data for NLS prediction, because 497-SMAKRAKKESQDF-508 was predicted only by cNLS Mapper as just a weak NLS activity, but not by other programs such as PSORT and PredictNLS.

Fig. 7. Comparison of NLS Sequences in 12 Drosophila Species

Multiple alignment was done with Clustal Omega (http://www.clustal.org/omega/). The residues of these proteins were colored according to their physicochemical properties: black, small amino acid residue; blue, acidic amino acid residue; red, basic amino acid residue; green, hydroxyl, sulfhydryl or amine amino acid residue. Asterisks indicate positions having a single, fully conserved residue; and colons, conservation between groups having strongly similar properties. The period indicates conservation between groups having weakly similar properties. D. mel, Drosophila melanogaster (accession NP_729983.1); D. sec, Drosophila sechellia (XP_002030494.1); D. sim, Drosophila simulans (XP_002084863.1); D. ere, Drosophila erecta (XP_001972738.1); D. yak, Drosophila yakuba (XP_002094796.1); D. ana, Drosophila ananassae (XP_001956407.1); D. pse, Drosophila pseudoobscura (XP_001353701.2); D. per, Drosophila persimilis (XP_002027023.1); D. moj, Drosophila mojavensis (XP_002009020.1); D. vir, Drosophila virilis (XP_002047885.1); D. wil, Drosophila willistoni (XP_002074414.1); and D. gri, Drosophila grimshawi (XP_001984985.1).

Three isoforms of the RecQ5 gene in Drosophila and humans are generated by differential splicing (DmRecQ5-RA, 470 aa; DmRecQ5-RB, 1058 aa; and DmRecQ5-RC, 468 aa,²²⁾ and HsRECQL5alpha, 410 aa; HsRECQL5beta, 991 aa; and HsRECQL5gamma, 435 aa,²¹⁾ respectively). Two of these, nearly identical 54-kDa proteins, consist of the helicase core only. As was shown in Fig. 3A, RecQ5 (aa 1–aa 468) would be similar to the small isoforms. These data suggest that the small isoforms of RecQ5 existed in the cytosol. The 121-kDa isoform migrates to the nucleus and exists exclusively in the nucleoplasm, whereas the small RecQ5 proteins stay in the cytoplasm.²¹⁾ Our data support the nuclear localization of the large isoform and cytoplasmic localization of the small isoforms, because only the large isoform has the NLS.²¹⁾

The nuclear import and export of macromolecules depends on energy and receptor-associated processes.²⁴⁾ Small proteins (<40 kDa) can cross the nuclear pore complexes by simple diffusion (e.g., EGFP of 26.9 kDa; Fig. 6B). However, in the case of large molecules, consensus sequence elements are required for trafficking in and out of the nucleus.²⁵⁾ It is reasonable that the RecQ5 protein, as well as BLM and WRN, would be transported into the nucleus, since this helicase catalyzing the unwinding of double-stranded DNA to provide single-stranded templates would be required in the nucleus. The NLS as having bipartite array of basic amino acids, RKRKKMPASQRSKRKK (aa 1334–aa 1349) and KRRCFPGSEEICSSSKRSK (aa 1371–aa 1389) are identified in the C-terminal of BLM²⁶⁾ and WRN,²⁷⁾ respectively. The nuclear transport of RecQ is important for its function as described in the case of WRN.²⁷⁾ A potential bipartite type of NLS with the sequence KRPRSQQENPESQPQKRPR exists in the C-terminus of human RECQL5.²¹⁾ However, Drosophila RecQ5 showed no similar amino acid sequence in the corresponding region (Fig. 2B). The classical NLSs rich in basic amino acids are known as NLSs recognized by importin α, and are classified into 2 major classes, monopartite and bipartite. Monopartite NLSs contain a single cluster of basic residues and are divided into 2 subclasses, one with at least 4 consecutive basic amino acids (class 1) and the other with 3 basic amino acids, represented by K(K/R)X(K/R) as a putative consensus sequence, where X indicates any amino acid (class 2). However, the NLS in Drosophila RecQ5, SHSSMAKRAKKESQDFI, would be a monopartite NLS and correspond to class 2. Thus, Drosophila RecQ5 NLS is unique in structure compared with NLS motifs found in other members of the RecQ helicase family, though its NLS resides in the C-terminal region.

Previously, we reported that the loss of maternally-derived RecQ5 leads to spontaneous mitotic defects in syncytial embryos.¹²⁾ These mitotic defects are derived from anaphase DNA bridges, which link pairs of daughter nuclei. These nuclei concurrently exit from the cycle and are eliminated by Drosophila checkpoint kinase 2 tumor suppressor homolog (DmChk2)-dependent centrosome inactivation. DmChk2 responds to DSB DNA lesions. These results suggest that the lack of RecQ5 led to spontaneous DSBs. RecQ5 may function in the resolution of anaphase DNA bridges during mitosis or in DSB repair during interphase in syncytial Drosophila embryos. In syncytial embryos, maternally -drived EGFP-RecQ5 was localized only in S-phase nuclei, and spread across over embryo in mitosis (Fig. 1), suggesting that the function of RecQ5 took place in the interphase. RecQ5 may function in DSB repair during interphase in syncytial Drosophila embryos. RecQ5 did not associate with mitotic chomosomes (Fig. 1C, supplemental movie). RecQ5 may not function in the resolution of anaphase DNA bridges during mitosis. However, the fine sub-cellular distribution of RecQ5 was not determined at high resolution. RecQ5 may associate with such an ultra-fine DNA bridge structure. The novel class of anaphase bridges, in which BLM, PICH, and FA proteins are involved, are prevalent in the anaphase population of normal human cells, and are present at an elevated frequency in cells lacking BLM.^28,29)

In conclusion, the RecQ5 protein was translocated into the nucleus by the NLS in its C-terminal region. This C-terminal region is known to be used for the regulation of RecQ5 function in both humans and Drosophila.^6–10,14) The NLS of Drosophila RecQ5 was distinct from that of human RECQL5 in terms of both position and amino acid sequence. On the other hand, Drosophila BLM has a putative bipartite NLS, RAGKRKKIYKSGASKRYK (aa 1414–aa 1431). The NLS of BLM is similar between humans and Drosophila with respect to both position and amino acid sequence.²⁶⁾ The NLSs of Drosophila nuclear proteins are sometimes distinct from those of human orthologues in their positions and amino acid sequences, as reported to be the case for G9a histone H3 methyltransferase.³⁰⁾ The nuclear transport of RecQ5 protein would be required basically for all organisms but would have evolved diversely in humans and Drosophila. The nuclear localization of RecQ5 by NLS would be important for RecQ5 function as well as for the function of other proteins, including Rad51.¹⁴⁾

Acknowledgment

This work was supported by the Science Research Promotion Fund from the Promotion and Mutual Aid Corporation for Private Schools of Japan. We thank Eishi Funakoshi, Kenji Takeuchi, and Katsuhisa Tashiro for many helpful discussions and technical support.

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