The DNA helicases are classified into five different superfamilies (SF1-SF5) (15). SF1 and SF2 helicases encompass a large group of DNA and RNA helicases found in eubacteria, archaea, viruses and eukaryotes. They possess an ATP-dependent translocation module consisting of two RecA-fold domains responsible for nucleic acid and ATP binding (15,16). HELQ was first described by Marini in 2002, which belongs to the SF2 protein family (1). SF2 family proteins have a pair of RecA-like domains that provide motion associated with helicase activity (6,17–19) and play key roles in chromatin rearrangement, DNA repair and transcription (20–22) and RNA metabolism (17,23). SF2 family proteins are divided into Ski2-like, RecQ-like, RecG-like, RecA-like ATP-dependent recombinase 3 (Rad3)/XPD, type I restriction enzymes, DEAD-box and NS3/NPH-II subfamilies, Swi/Snf, DEAH/RNA helicase A, RIG-I-like, based on sequence homology (6,24,25). In archaea, in all Ski2-like helicases including Mtr4, Ski2, and Bad Response to Refrigeration 2 homolog, the molecular ‘core’ of them is a ring-like assembly of four domains comprising a ratchet domain, a winged helix domain and two RecA domains (25).

HELQ maps to chromosome 4q21 (1) and generates a full-length mRNA (NM_133636.5) comprising 3,543 base pairs and encoding a protein of 1,101 amino acids (aa) (1). A total of 7 transcribed splice isoforms of HELQ have been described, encoding six HELQ protein variants (26). HELQ is a conserved protein that possesses three main protein domains: The DEAD/DEAH box helicase domain, a helix-turn-helix (HTH_61) domain and a helicase C-terminal domain (Fig. 1). The domain of DEAD/DEAH box helicase contains a DEAH box and an ATP-binding region (putative ATP binding site), and is involved in unwinding nucleic acids. In addition, human HELQ has a disordered domain (212–261 aa), containing two compositionally biased regions, comprising basic and acidic (214–228 aa) and polar (229–253 aa) residues. A structural diagram of the human HELQ protein is presented in Fig. 1.

The DEAD/DEAH box helicase class of proteins, which share eight conserved motifs (I, Ia, Ib, II, III, IV, V and VI) (27). ATP is bound to motifs I and II, while RNA is bound to motifs Ia and Ib. The DEAD/DEAH box is named after the Walker B motif (motif III) consensus sequence (28) and functions in ATP hydrolysis. Motifs IV and V perform similar functions to those of motifs Ia and Ib, respectively, in RNA binding (27). DEAD/DEAH box helicases are found in various prokaryotes and eukaryotes (28), and are involved in several aspects of RNA metabolism, such as pre-mRNA splicing, RNA decay, nuclear transcription, editing, nucleocytoplasmic transport, ribosome biogenesis, translation and organellar gene expression (29–31).

The helicase C-terminal domain (HelicC) is present in various SF1 and SF2 helicases and helicase-related proteins. The HelicC domain does not fold autonomously, but rather as a component of the helicase, which participates in the ATP-dependent unwinding of DNA or RNA.

The α-helical protein domain family includes winged helix-turn-helix (HTH) domains that have characteristic folds, which function as sequence-specific DNA-binding domains (32). The HTH plays an important role in DNA binding and protein interactions (33,34) and consists of two α-helices (α-helix 22 and 23) and a β-sheet turn, where a double-stranded DNA (dsDNA) major groove can be recognized by α-helix 23 (14,2).

HELQ is highly conserved across a wide range of species from archaea through to mammals (https://www.ncbi.nlm.nih.gov/homologene/?term=HELQ). For example, Homo sapiens HELQ shares a >97, 78 and 60% DNA similarity with the homologous Macaca mulatta, Mus musculus, and Danio rerio genes, respectively (Table I). It is a conserved gene in eukaryotes that HELQ (HomoloGene ID: 14667) has important molecular functions (i.e., ATP binding, DNA binding, single-stranded 3′-5′DNA helicase activity) and cellular component classifications (i.e., nucleus, site of DNA damage), and acts in biological processes (i.e., DNA duplex unwinding, rRNA processing) (1,9,35–37) (https://www.alliancegenome.org/gene/HGNC:18536) (Table II). Since HELQ is expressed in a wide variety of species, it was likely present in a common ancestor of vertebrates.

DNA damage is a common event that may have either endogenous or exogenous causes and can lead to mutations, cell/organ death and cancer (39,40). It is crucial to repair damaged DNA after it has been damaged, as this allows damaged DNA to regain its original structure and function normally (34,41). Various pathways recognize and repair different types of DNA lesions, including direct repair, NER, base excision repair, inter-strand cross-link (ICL) repair and double-strand break (DSB) repair (39). HELQ maintains genomic stability and avoids tumorigenesis through its involvement in different repair pathways, including NER, DSB repair and ICL repair (9,11–13).

A NER pathway is involved in the removal of DNA damage after certain types of DNA damage have occurred (13). The expression levels of NER pathway proteins [e.g., xeroderma pigmentosum complementation group A (XPA), XPC, replication protein A (RPA) and ERCC excision repair 1, endonuclease non-catalytic subunit] are important mediators for responses to platinum-based chemotherapy and influence DNA repair activity (13,42). HELQ is crucial for cellular responses to cisplatin through its role in regulating the expression of NER pathway proteins (13).

DSBs are major DNA lesions deleterious to cell survival and genetic stability (43). When DSBs are not repaired, they can result in chromosome loss and rearrangements and even carcinogenesis (44). Two major pathways can repair DSBs: Homologous recombination (HR) and non-homologous end joining (NHEJ) (45,46), as well as alternative pathways, such as microhomology-mediated end joining (MMEJ) and single strand annealing (SSA) (45,46). HR is essential for DSB repair in post-replicative chromatin following replication fork collapse (47,48), and requires the loading of the RAD51 recombinase onto single-stranded DNA (ssDNA) through DNA ends or at post-replicative ssDNA gaps (45). The function of HELQ in HR is to capture RPA-bound ssDNA and then to displace it to speed up the annealing of complementary DNA strands (9). HELQ immediately interacts with BCDX2, a paralog of RAD51, to accelerate effective HR in damaged replication forks (11). RFS-1 binds to HELQ and plays a complementary role in facilitating the breakdown of RAD51 dsDNA filaments in postsynaptic HR intermediates, which is required to complete meiotic DSB repair in Caenorhabditis elegans (47). In addition, the HR factor with OB-fold (HROB)-mini-chromosome maintenance 8 (MCM8)-MCM9 pathway functions redundantly with HELQ, supporting a postsynaptic step of HR in a parallel pathway (49). HELQ accelerates HR-efficiency at compromised replication forks by working in parallel with the FA pathway in HELQ^ΔC/ΔC and FANCD2^−/− double mutant mice (11). Furthermore, HELQ is essential for the function of the synthesis-dependent strand annealing modes of HR, MMEJ of G4-induced DSBs and SSA in genome stability and tumor avoidance (9).

ICLs are also deleterious DNA lesions caused by endogenous (malondialdehyde) or exogenous [mitomycin C (MMC); cis-platinum and psoralens] sources (50,51), which induce mutations and chromosomal rearrangements by inhibiting DNA replication and transcription (1,34). ICL repair is complex and proteins from the NER, translation synthesis and HR pathways are involved in ICL repair (40). With its association with RAD51 paralogs, HELQ prevents germ cell loss and reduces cancer susceptibility through ICL repair (11,52). HELQ deficiency leads to germ cell attrition and ICL repair sensitivity in mouse and human cells (11,12,53). The FA pathway plays a critical role in recruiting RAD51-mediated HR during ICL repair (40). In parallel with HELQ, HROB participates in ICL repair as epistatic with MCM8-MCM9 (49), which also directly facilitates the repair of ICL-induced damage downstream of FANCD2 ubiquitylation (49,54).

The HELQ HTH domain is associated with DNA strand binding and protein interactions (2,34). The dissociation constant values of wild-type HELQ for ssDNA and dsDNA were reported as 0.14 and 5.3 µM, respectively, indicating a strong preference for ssDNA binding and suggesting that the protein must track along, displacing the dsDNA strand (2). Furthermore, HELQ mutations reduce dsDNA binding, but ssDNA binding is not affected (55). The HELQ-ssDNA interaction is essential for the translocation mechanism. Mechanistically, HELQ interacts with RPA, and RPA coordinates the loading of HELQ onto ssDNA. HELQ helicase core is activated by ATP-Mg²⁺ binding and translocates along ssDNA as a dimer when loaded onto ssDNA (35).

In addition, HELQ is an ssDNA-activated ATPase, which is important for unwinding forked DNA (10,56). As well as unwinding ssDNA and dsDNA junctions, and HELQ is capable of unwinding 3′ overhangs, 3′ lagging strand forks, Y-structures, and D-loops (3); however, HELQ cannot unwind using ATPγS or 5′ overhang substrates (9). The HELQ unwinding of 3′ overhang substrates is inhibited by RPA, whereas RAD51 stimulates the unwinding activity of its D-loops by forming a complex with HELQ (9).

HELQ is an ATP-dependent enzyme involved in the recovery of replication forks that have stalled or collapsed following DNA damage (3). HELQ can act on damaged replication forks where the leading strand template for DNA replication has stopped, causing the polymerase to uncouple and continue DNA synthesis from the lagging strand template (10). Following treatment with camptothecin (CPT), an agent that stalls and collapses replication forks, HELQ is recruited to stalled replication sites that are associated with replication resumption. HELQ can facilitate replication resumption, possibly through colocalizing with IdU incorporation sites and RPA foci, by unwinding the nascent lagging strand, or alternatively through HELQ co-localization with RAD51 and FANCD2 at sites of stalled replication (3,12). In addition, HELQ has an important role in rescuing stalled forks during the normal S phase, and the loss of HELQ results in increased stalled forks, a role which is not epistatic with that of FA complementation group C (53).

Previous studies of HELQ have focused more on its DNA repair and DNA unwinding functions, while its role in DNA strand annealing has been underappreciated. Tafel et al (3) reported that HELQ does not exhibit a strong annealing activity, and that RPA can suppress separated strand reannealing by binding to unwound ssDNA generated by HELQ. By contrast, Anand et al (9) reported that RPA strongly accelerated the DNA strand annealing activity of HELQ. Mechanistically, in addition to capturing RPA-bound DNA strands, HELQ is capable of superseding RPA and stimulating complementary DNA strands. Furthermore, it was found that ATP binding and hydrolysis are essential for HELQ DNA annealing activity, whereas the RAD51 addition was unaffected by HELQ-dependent DNA annealing activity (9).

CHK1 is a primary effector of DNA lesion and replication checkpoint responses, and its inhibition promotes DNA damage and reduces HR repair (57,58). CHK1 physically interacts with RAD51, while CHK1/RAD51 disruption or inactivation induces defective HR, aberrant replication dynamics, and chromosome instability (59). In osteosarcoma cells, CHK1 activation is promoted by HELQ, and HELQ colocalizes with RAD51 to participate in the repair of damaged forks by HR (3).

FA is a rare genetic disease, and patients with FA are hypersensitive to DNA ICL-inducing agents such as mitomycin C and cisplatin (60). FA signal transduction involves 22 proteins, which share a common pathway that is activated on DNA damage (61). FANCD2 is currently the focus of research into the FA pathway, and is crucial in cellular responses to DNA lesions (62). HR may be regulated by the FA pathway, where activated FANCD2 is translocated to chromatin-associated foci, where it colocalizes with HR proteins [RAD51 and breast invasive carcinoma (BRCA)2] (63). The strong negative effect of HELQ knockdown on single-stranded templated repair via the HR repair pathway may be explained by its physical interaction with FANCD2 and RAD51 (64). Following CPT treatment, HELQ is involved in fork repair and restart by localizing to replication forks, unwinding lagging strand structures, and co-localizing with RAD51 and FANCD2 (3,14). In addition, although the FA pathway, ICL sensitivities, and HELQ are additive, there is no epistasis of HELQ for downstream targets of FANCD2, and HELQ plays an independent role from FA pathway in ICL processing (12).

RAD51 is a DNA-binding protein that can bind to both ssDNA and dsDNA and maintain genome stability in DNA replication (65,66). RAD51 is also an ATPase that forms nucleoprotein filaments on ssDNA and facilitates the search for homologous repair templates, such as homologous chromosomes, sister chromatids or ectopic homologous sequences (67). Hence, RAD51 is essential for regulating fork reversal by discovering and invading homologous DNA sequences during DSB repair by HR (65,67). In addition, five RAD51 paralogs [RAD51B/C/D and X-ray repair cross complementing (XRCC)2/3] that form two main complexes, BCDX2 (RAD51B/C/D-XRCC2) and CX3 (RAD51C-XRCC3), are involved in the HR repair of collapsed replication forks and maintenance of genome stability (68–70). The BCDX2 complex preferentially binds to ssDNA and accelerates HELQ ATPase activity (71), which plays a central role in recognizing damage and stimulating fork remodeling in response to fluctuating dNTP pools (72). Adelman et al (11) found that HELQ plays a critical role in replication-coupled HR by interacting with complexes of the RAD51 paralog, BCDX2, to avoid germ cell loss and tumorigenesis. Anand et al (9) also demonstrated that HELQ binds to BCDX2 complexes, and that the latter strongly stimulates the translocation of HELQ during DNA unwinding. Takata et al (12) reported that HELQ interacts with ATR and HR-related RAD51 paralogs and participates in DNA LCL resistance in human cells.

As a major ssDNA binding protein, RPA is abundant in eukaryotic cells (66,73). RPA comprises three subunits, RPA14, RPA32 and RPA70, which are first responders in the event of the disruption of DNA metabolism. RPA is essential for replication, recombination and repair by binding to ssDNA to maintain genome duplication and stability (74). RPA and HELQ physically interact to form a supershift complex with HELQ on ssDNA (4). Furthermore, RPA stimulates HELQ helicase activity by binding to unwound regions generated by HELQ and inhibiting reannealing (4). Similarly, in mammals, RPA can restrain DNA unwinding while promoting DNA strand annealing by HELQ. Mechanistically, HELQ is recruited to ssDNA by interacting with RPA, followed by RPA displacement to stimulate complementary DNA strand annealing (9).

HELQ plays a critical role in tumor suppression in mammals through interacting with the BCDX2 complex (11). HELQ overexpression inhibited osteosarcoma cell proliferation, migration, invasion and DNA damage repair (86). In addition, the antitumor activity of HELQ may be associated with the upregulation of the DNA damage-related proteins RAD51 and CHK1expression, and HELQ modulates an anti-invasive phenotype and DNA damage repair in osteosarcoma cells by activation of the CHK1-RAD51 signaling pathway (86).

HELQ is downregulated in NSCLC tissues and cells, while the malignancy of lung cancer cells was enhanced by HELQ depletion. HELQ overexpression inhibits cell proliferation and migration by suppressing DNA damage repair, and promotes cell death by inducing necrosis through its interaction with receptor-interacting serine/threonine kinase 3 (RIPK3) (89). HELQ is a favorable prognostic factor for patients with NSCLC, and low HELQ levels in patients with NSCLC were associated with a reduced overall survival (OS) through a Kaplan-Meier plotter.

HELQ and RAD51C expression levels are decreased in ESS compared with normal endometrial tissues. HELQ expression was found to be correlated with the size and type of ESS. Neither HELQ nor RAD51C expression were correlated with age, FIGO stage or lymph node metastasis status. The occurrence and development of ESS may be affected by DNA repair involving HELQ and RAD51C (90).

In addition, HELQ plays a role in cellular resistance in response to ICLs and G2/M arrest, through its association with the RAD51 paralogs RAD51B/C/D and XRCC2, and promotes the activation of the ATR substrate, CHK1 (12). HELQ and XPA binding protein 2 are associated with platinum resistance, poor prognosis, decreased apoptosis and increased DNA damage repair in ascites from high-grade serous OV cells (87). In addition, HELQ decreases cisplatin sensitivity in OV cells by activating the NER and ATM/ATR pathways (13). OV patients with HELQ-low expression had improved overall and disease-free survival than those with a high HELQ level. There is evidence that HELQ can be used independently as a prognostic marker to predict survival in patients with OV (13).

CLL patients with low HELQ levels exhibited a significantly unfavorable OS compared with patients with high HELQ levels. HELQ may be useful in predicting patients at high risk for CLL based on the Richter transformation. Higher HELQ expression was also associated with an improved response to immuno-chemotherapy in patients with CLL. HELQ represented a prognostic marker for CLL associated with the activation of MYC signaling, E2 factor signaling and DNA repair pathways, as well as the suppression of Hedgehog and Kras signaling (88).