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Introduction

Ovarian cancer is the second most common gynecological cancer worldwide, accounting for ~3% of all female cancer cases, with the typical age of diagnosis being 63-years-old. It is challenging to diagnose ovarian cancer at an early stage, and this cancer has a poor prognosis, with a five-year survival rate of ~47% (1). In order to provide a foundation for detecting and treating ovarian cancer, studies investigating the etiology of the disease are required. Previously, a study by the London Research Institute (Cancer Research UK, London, UK) found helicase, POLQ-like (HELQ) to be a novel gene that prevents ovarian cancer (2). In this study, the possibility of ovarian cancer in mice was increased two-fold when a copy of the HELQ gene was missing. Even the deficiency of one copy could lead to the formation of an increased number of tumors in mice. According to this study, detection of HELQ gene deficiency may be adopted to screen for ovarian cancer patients among women in the future, if HELQ helicase performs the same role in humans and mice (2).

HELQ is a superfamily II DNA helicase that was first identified in the human and mouse genomes through its homology to mutagen-sensitive 308 (Mus308) (3), a DNA repair enzyme required for DNA interstrand crosslink (ICL) resistance in Drosophila melanogaster (4,5). DNA ICLs are particularly toxic as they disrupt genetic information on strands, potently inhibiting DNA replication and transcription. In normal cells, DNA ICLs and DNA damage often lead to the occurrence of cancer (6). However, DNA repair is an important reaction following DNA damage, which may cause the damaged DNA to revert to the original appearance and perform the original function (7). DNA helicases play an important role in this process. HELQ, as a DNA helicase, has been studied from the perspective of association with cancer. Previous genome-wide association studies have identified single nucleotide polymorphisms at loci within or near the HELQ gene that are associated with an increased risk of several different cancers, including upper aerodigestive tract cancers and head and neck cancers (8–11).

To assess the association between the structure of HELQ and the carcinogenesis of ovarian cancer, bioinformatics methods were used to analyze this association from a theoretical angle in the present study. It was expected that such a study may provide direction and basis for the clinical treatment of ovarian cancer.

Materials and methods

Gene sequence

The HELQ gene sequence was obtained from the Nucleotide resource of the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/nucleotide), using the GenBank accession number AF436845.1 and Gene ID 113510. The HELQ gene is also termed HEL308.

Bioinformatics analysis

The BioEdit software (http://www.mbio.ncsu.edu/bioedit/bioedit.html) was used for the analysis of open reading frames. The ExPASy tools ProtParam (http://web.expasy.org/protparam/) and ProtScale (http://web.expasy.org/protscale/) were used to compute gene features, including the amino acid composition, molecular mass, isoelectric point, hydrophobicity/hydrophilicity, instability index and aliphatic index. The SignalP-HMM Server was used to predict the signal peptides (12). The transmembrane region was analyzed using the TMHMM Server V.2.0 system (http://www.cbs.dtu.dk/services/TMHMM/). Subcellular localization was predicted using the Protein Subcellular Localization Prediction Tool (PSORT) database (http://www.psort.org/). The Simple Modular Architecture Research Tool (SMART; http://smart.embl-heidelberg.de/) was used for analysis of protein domains. Hopfield Neural Network (HNN) was used to make the secondary structure predictions (13). PHYRE2 Protein Fold Recognition Server online software (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) was employed to obtain the three-dimensional structure of HELQ and the 3DLigandSite Server (http://www.sbg.bio.ic.ac.uk/3dligandsite/) was used to construct the three-dimensional ligand binding model. STRING9.0 interactive database (http://string-db.org/) was utilized to find the associated proteins. Gene ontology analysis was performed using PredictProtein software (https://www.predictprotein.org/).

Results

Analysis of physicochemical properties of HELQ

Through NCBI database retrieval, the whole nucleotide sequence and amino acid sequence of HELQ were obtained, with a total sequence of 3,591 bp encoding 1,101 amino acid residues. Open reading frame (ORF) prediction and BioEdit analysis showed that the HELQ gene contained 15 ORFs. ProtParam predicted that the molecular weight of the HELQ gene was 124,175.3 Da, the theoretical isoelectric point was 6.12, the content of leucine (Leu) was 16.2% of the total components, acidic amino acids were more common than basic amino acids, and the instability index was 45.55. Therefore, it was inferred that HELQ was an unstable and acidic protein. In addition, the value of grand average of hydropathicity was −0.317 and the aliphatic index was 92.34, which indicated that HELQ is liposoluble.

Subcellular localization and motif prediction

The location of proteins in cells is closely associated with the function of proteins. Subcellular localization analysis of PSORT prediction showed that HELQ was mainly distributed in the nucleus, mitochondrial matrix space, microbody (peroxisome) and mitochondrial inner membrane (Table I). HELQ was located in areas with the presence of DNA.

Table I. Subcellular localization of helicase, POLQ-like encoding product.

Table I.

Subcellular localization of helicase, POLQ-like encoding product.

Subcellular localization	Certainty
Nucleus	0.600
Mitochondrial matrix space	0.510
Microbody (peroxisome)	0.300
Mitochondrial inner membrane	0.234

As for motifs, the functional structure domain database of ScanProsite predicted that the HELQ protein contained two functional domains, consisting of Helicase_ATP-Bind_1 and Helicase_Cter, and these domains were responsible for binding and hydrolyzing ATP, respectively, which conformed to the characteristic of DNA helicase depending on ATP hydrolysis (Fig. 1A). Domain architecture analysis using SMART showed that 5 main domains could be found in the HELQ sequence, with other features not shown in the diagram (Fig. 1B) due to overlap. By searching the NCBI Conserved Domains Database, 4 domains were found, which were DEXDc, HELICc, HHH-5 and PRK02362 (Fig. 1C). According to the prediction of SignalP-HMM and TMHMM, the HELQ protein has no evident signal peptide and transmembrane domain.

Figure 1. Prediction of functional domains. (A) ScanProsite; (B) Simple Modular Architecture Research Tool; (C) National Center for Biotechnology Information conserved domains.

Structural analysis of the HELQ protein

HNN was used to analyze the secondary structure of the HELQ protein. Results showed that HELQ was mainly composed of α-helixes (46.48%) and random coils (43.05%), with extended strands accounting for only 10.26% of the structure (Fig. 2). According to the prediction of the tertiary structure using the threading method in the PHYRE2 Protein Fold Recognition Server, the best template protein was c2va8A (Protein Data Bank code), which had high homology with HELQ (Fig. 3). The 3DLigandSite Server was used to construct the three-dimensional ligand binding model (Fig. 4). It was found that the ligand binding sites were distributed over ILE333, LYS335, TYR337, GLN340, SER362, GLY363, GLY364, LYS365, THR366, LEU367, LYS397 and ASN678. Heterogens present in the predicted binding sites consisted of 10 adenosine diphosphate (ADP), 14 Mg2+ (MG) and 3 adenosine monophosphate (AMP). The functional domains Helicase_ATP-Bind_1, Helicase_Cter and HHH_5 were separated from the HELQ sequence and were respectively analyzed by building ligand binding models. The results suggested that ligand binding sites in the Helicase_ATP-Bind_1 domain distributed over SER17, GLY18, GLY19, LYS20, THR21, LEU22, LYS52 and the heterogens present in the predicted binding sites contained 12 ADP, 14 MG and 3 AMP. The binding site present in the Helicase_Cter domain was ASN113, and the heterogens contained 4 ADP, 12 MG and 1 ATP. The binding site in the HHH_5 domain was GLU15 and the heterogens contained 7 MG and 33 Ca2+. The activity of HELQ is dependent on the binding of ATP by the Helicase_Cter domain to supply energy. When functional domains of HELQ are not complete, DNA replication cannot progress normally.

Figure 2. Secondary structure of the helicase, POLQ-like protein.

Figure 3. Tertiary structure of the helicase, POLQ-like protein.

Figure 4. Construction of three-dimensional ligand binding models of (A) helicase, POLQ-like, (B) Helicase_ATP-Bind_1 domain, (C) Helicase_Cter domain and (D) HHH_5 domain.

Prediction of protein-protein interactions

STRING9.0 interactive database was utilized to determine the functional protein association networks. Proteins that interact with HELQ mainly included regulator of telomere elongation helicase 1 (RTEL1), family with sequence similarity 175 member A (FAM175A), small ubiquitin-like modifier 1 (SUMO1), DNA polymerase ν (POLN) and coiled-coil domain containing 158 (Fig. 5). They were coexpressed and had similar functions in repairing the DNA lesions appearing in the process of DNA replication during cell proliferation.

Figure 5. Cross-linked analysis of HELQ protein. RTEL1, regulator of telomere elongation helicase 1; FAM175A, family with sequence similarity 175 member A; HELQ, helicase, POLQ-like; CCDC158, coiled-coil domain containing 158; SUMO1, small ubiquitin-like modifier 1; POLN, DNA polymerase ν.

Gene ontology analysis

Gene ontology analysis was performed using PredictProtein software. Molecular function ontology showed that the activity of HELQ mainly consisted of protein binding, and helicase, ATP-dependent RNA helicase, DNA strand annealing, ATPase, ATP-dependent helicase, RNA helicase and RNA-dependent ATPase activity (Table II). In addition, biological process ontology analysis indicated that HELQ mainly participated in the process of DNA repair (Table III).

Table II. Molecular function ontology.

Table II.

Molecular function ontology.

GO ID	GO term	Reliability, %
GO:0005515	Protein binding	26
GO:0004386	Helicase activity	17
GO:0004004	ATP-dependent RNA helicase activity	14
GO:0000739	DNA strand annealing activity	14
GO:0016887	ATPase activity	14
GO:0008026	ATP-dependent helicase activity	12
GO:0003724	RNA helicase activity	11
GO:0008186	RNA-dependent ATPase activity	10
GO:0042802	identical protein binding	6
GO:0043140	ATP-dependent 3′-5′ DNA helicase activity	4
GO:0043621	Protein self-association	4
GO:0005524	ATP binding	4
GO:0043008	ATP-dependent protein binding	4
GO:0008432	JUN kinase binding	3
GO:0017151	DEAD/H-box RNA helicase binding	3
GO:0003678	DNA helicase activity	3
GO:0030621	U4 snRNA binding	2
GO:0003712	Transcription cofactor activity	2
GO:0017070	U6 snRNA binding	2
GO:0003743	Translation initiation factor activity	2
GO:0003730	mRNA 3′-UTR binding	2
GO:0003677	DNA binding	2
GO:0000339	RNA cap binding	2
GO:0003723	RNA binding	2
GO:0008143	Poly(A) RNA binding	2
GO:0008094	DNA-dependent ATPase activity	1
GO:0033592	RNA strand annealing activity	1
GO:0000403	Y-form DNA binding	1

[i] GO, gene ontology; ATP, adenosine triphosphate; JUN kinase, c-Jun N-terminal kinase; snRNA, small nuclear RNA; UTR, untranslated region.

Table III. Biological process ontology.

Table III.

Biological process ontology.

GO ID	GO term	Reliability, %
GO:0006281	DNA repair	28
GO:0006310	DNA recombination	9
GO:0006397	mRNA processing	9
GO:0006364	rRNA processing	9
GO:0009615	Response to virus	8
GO:0045449	Regulation of transcription	8
GO:0006314	Intron homing	8
GO:0006268	DNA unwinding involved in replication	8
GO:0000398	Nuclear mRNA splicing, via spliceosome	8
GO:0008380	RNA splicing	8
GO:0040007	Growth	8
GO:0006260	DNA replication	8
GO:0007126	Meiosis	8
GO:0006350	Transcription	8
GO:0009792	Embryonic development ending in birth or egg hatching	8
GO:0006417	Regulation of translation	8
GO:0000393	Spliceosomal conformational changes to generate catalytic conformation	8

[i] GO, gene ontology.

Discussion

DNA helicases have crucial roles in maintaining genome stability and stable DNA replication in all organisms. They have been implicated in nucleotide excision repair, mismatch repair, base excision repair, double strand break repair and cross-link repair (14). The helicases PriA, RecG, RuvAB, RecBCD, UurD, Srs2, Rep and RecQ are well known for roles in promoting DNA repair and recombination by several possible mechanisms (15–17). Similar to RecQ, the Mus308 family of helicases supports genome stability. The Mus308 locus was identified in Drosophila melanogaster, being required for resistance to DNA crosslinking agents (4). Moldovan et al (18) found that the deletion of HEL308 in human cells could induce sensitivity to replication-blocking lesions, namely the ICLs induced by mitomycin C and irinotecan hydrochloride. Biochemical studies have established that human HEL308 is an ATP-dependent enzyme that unwinds DNA with a 3′-5′ polarity (3,19), which is consistent with the structure of HELQ containing ATP-binding sites found in the present study. In the functional domains of HELQ, Helicase_ATP-Bind_1 and Helicase_Cter locate in the center position and they mainly perform helicase activity. At the C-terminal, the helix-hairpin-helix configuration may be important for binding to DNA strands. The results of the functional protein association networks found that HELQ was involved in ubiquitination in the process of DNA repair. RTEL1 is responsible for the maintenance of chromosome ends and has a synergistic effect with proliferating cell nuclear antigen (PCNA) in the process of DNA replication to ensure cell growth and division, and to avoid genetic mistakes. When RTEL1 cannot combine with PCNA, DNA replication cannot continue and the errors produced lead to cancer (20). FAM175A mediates the formation of the BRCA1-RAP80 complex, which adds or modifies existing ubiquitin chains to promote DNA damage repair (21). SUMO1 is important in the control genomic integrity. Hu et al (22) found that SUMO1 repaired DNA lesions through an ubiquitin-like modification path. POLN works as a DNA polymerase and participates in DNA repair to make DNA replication proceed normally (18). However, the exact mechanism of HELQ remains unclear.

An increasing number of studies focus on the function exploration of HELQ. Ward et al (23) found that HELQ-1 played a role in meiotic double-strand break repair by promoting postsynaptic RAD-51 filament disassembly in Caenorhabditis elegans. Another study identified that in humans, HELQ was expressed in the ovaries, testes, heart and skeletal muscle (24). Luebben et al (25) reported that mammalian HELQ contributed to genome stability in unchallenged conditions through a mechanism distinct from the function of Fanconi anemia complementation group C. HELQ in humans requires additional investigation, particularly the association with cancer. The occurrence of tumors is usually closely associated with abnormal DNA replication and cell proliferation. Appropriate readjustment in the structure of HELQ to change its primary function may be used to alleviate DNA damage or promote DNA variation.

The genetic complexity of cancer has posed a challenge for devising successful therapeutic treatments. Tumor resistance to cytotoxic chemotherapy drugs and radiation, which induce DNA damage, has limited their effectiveness (26). Targeting the DNA damage response is one strategy for combating cancer. The prospect for success of chemotherapy treatment may be improved by the selective inactivation of a DNA repair pathway.

In conclusion, the structure of HELQ protein was predicted and analyzed in this study. Its unique structural characteristics will have an important role in future investigations of HELQ gene deletion, as well as the etiological analysis and targeted therapy of ovarian cancer.

References