Helicases at Work: The Importance of Nucleic Acids Unwinding Under Cold Stress

Abstract

Separation of duplex strands of nucleic acids is a vital process in the nucleic acid metabolism and survival of all living organisms. Helicases are defined as enzymes that are intended to unwind the double-stranded nucleic acids. Helicases play a prominent role in the cold adaptation of plants and bacteria. Cold stress can increase double-strand DNA breaks, generate reactive oxygen species, cause DNA methylation, and stabilize the secondary structure of RNA molecules. In this review, we discuss how helicases play important roles in adaptive responses to cellular stress caused by low temperature conditions, particularly in bacteria and plants. We also provide a glimpse of the eminence of helicase function over nuclease when an enzyme has both helicase and nuclease functions.

1. Introduction

In the current erratic weather patterns, all living organisms must learn to adapt and grow under various environmental stress conditions. To survive, living organisms must endure and evolve to grow under various adverse environmental conditions, such as high pressure, fluctuating pH, extended dry conditions, osmolarity changes, nutrient scarcity, and extremely low or high temperatures. Bacteria, in particular, exhibit remarkable adaptability, thriving in diverse temperature ranges from the permafrost bacterium Planococcus halocryophilus, which grows at −15 °C, to the hyperthermophilic Geothermobacterium ferrireducens, which thrives at 100 °C [1,2]. The ability of bacteria to grow across such a broad spectrum of temperatures is made possible through various cellular, molecular, biochemical, and physiological adaptations [3]. However, growth at low temperatures presents unique challenges. Cold environments reduce membrane fluidity, cause structural changes in proteins, decrease enzyme activity, and stabilize nucleic acid structures, hindering cellular functions [4]. To counteract these effects, microorganisms must implement numerous adjustments. They modify membrane composition to maintain fluidity, employ enzymes with enhanced structural flexibility, and optimize key processes such as DNA replication, transcription, and translation to sustain cellular metabolism under cold stress [3,5].

Plants being immobile, they must withstand harsh weather conditions to survive and grow. Plants can sense fluctuations in temperature through membrane fluidity, structural modification of nucleic acids and proteins, cytoskeleton assembly, and altered enzymatic activities [6]. Cold stress can seriously affect plant growth and development, causing reduced crop productivity. Therefore, understanding how plants detect and regulate cellular functions in response to cold stress is of great interest to researchers. Sensing and regulating the cold stress through cell membrane fluidity, secondary messengers [such as calcium ion (Ca²⁺), reactive oxygen species (ROS), nitric oxide (NO), superoxide, and hydrogen peroxide (H₂O₂)] have been considerably well investigated [7]. The transcriptional, post-transcriptional, and epigenetic regulation of gene expression under cold stress have also been well documented [7,8]. In this review, we focus on the crucial role of DNA and RNA helicases, particularly those from the SF1 and SF2 DEAD-box families, in low temperature adaptation. We highlight the importance of nucleic acid unwinding and its significance in cold stress response, adaptation, and acclimatization.

2. Helicases

The first nucleic acid-unwinding enzyme was discovered in 1976 [9,10,11]. Two years later, Hoffmann-Berling and colleagues coined the term “helicase” to define the ATP-dependent DNA unwinding property of proteins [12]. Since then, remarkable progress has been made in understanding the mechanistic and functional roles of helicases and their biological importance. Helicases are the nucleic acid-dependent ATP hydrolyzing enzymes that are capable of unwinding DNA or RNA duplex structures. Based on their sequence, characteristic motifs, structure, and mechanisms, helicases are categorized into six superfamilies (SF 1 to 6) [13]. The core domains and characteristic motifs of each superfamily are summarized in Figure 1A.

Based on the structural organization and domain arrangements, these superfamilies are further categorized into two major groups: SF1 and SF2, and SF3 to SF6. Helicases of the SF1 and SF2 families are monomers that structurally have a single polypeptide chain with two RecA domain-like folds (Figure 1B). On the other hand, the SF3 to SF6 family helicases are hexamers/double-hexamers containing six or 12 domains of either RecA-like folds or AAA+ (ATPases associated with various cellular activities) (Figure 1B). Helicases of these superfamilies perform a wide range of crucial cellular functions by moving along in 5′ → 3′ or 3′ → 5′ direction on various nucleic acid substrates (DNA, RNA, and DNA-RNA hybrids). In 2007, Wigley and co-workers proposed and classified helicases further into types A and B, and α- and β-helicases based on their direction of translocation on nucleic acid and substrate specificity, respectively [13]. As proposed, the helicases that translocate on a single-stranded (ss) DNA or ssRNA in the 3′ → 5′ direction are considered as A type and in the 5′ → 3′ direction as B type (Figure 1C). Similarly, helicase-like proteins that move along ssDNA/RNA or double-stranded DNA (dsDNA) in either direction (5′ → 3′ or 3′ → 5′) are classified as α- and β-helicases, respectively (Figure 1D). Importantly, both type A and B commonly require a 3′-terminated ssDNA flanking duplex DNA or a 5′-terminated ssDNA flanking duplex DNA to function as helicases. Many of them are not bona fide helicases but translocate by sliding on ssDNA or RNA coupled with ATP hydrolysis and separate duplex strands. For detailed information on the superfamilies of helicase, the reference [13] is recommended.

Superfamily 1 (SF1): SF1 is a family of structurally well-characterized proteins comprising a large number of DNA and RNA helicases. The SF1 helicases are defined by the presence of characteristic amino acid sequence motifs. SF1 family helicases have 13 specific motifs, including the Walker A (motif I), Walker B (motif II), and arginine finger (motif IV) motifs (Figure 1A). The highest level of sequence conservation is observed in the motifs I, II, and IV that coordinate nucleotide triphosphate binding and hydrolysis [14]. Three defined subfamilies have been identified so far within the SF1: UvrD/Rep-like, Pif1-like, and Upf1-like families (Figure 2) [14]. Most of the UvrD/Rep-like enzymes are SF1Aα helicases, whereas Pif1 and Upf1-like proteins are SF1Bα helicases. SF1 helicases act on both DNA and RNA substrates [15].

Superfamily 2 (SF2): SF2 is the largest of all the helicase superfamilies and is implicated in diverse cellular processes, including RNA and DNA metabolisms. Similar to SF1, SF2 helicases also contain a single polypeptide chain with two core RecA-like fold domains [13]. The SF2 family proteins share at least 12 characteristic amino acid sequence motifs with SF1 helicases (Figure 1A). Based on the alignment of the helicase cores of all the helicases, SF2 is further divided into nine families and one group [14]. These families include DEAD-box, DEAH/RHA, RecQ-like, Rad3/XPD, Swi/Snf, type I restriction endonucleases (type I RE), RIG-I-like, RecG-like, Ski2-like families, and the SN3/NPH-II group of proteins (Figure 2).

Superfamily 3 (SF3): The SF3 family includes helicases that are involved in the viral replication cycle, replication origin recognition, melting, and unwinding [16]. SF3 helicases contain AAA+-like fold helicase domains and organize into hexamers or double-hexamers to form toroidal structures (Figure 1A–C). Helicases in this family share five conserved motifs: A, B, B′, C, and R. The motif C is specific to SF3, and the conserved arginine finger (R) is located after motif C (Figure 1A). They translocate along nucleic acid in a 3′ → 5′ direction (type A enzymes) (Figure 1C) [13]. The best-studied members of this family are the large T antigen from the simian virus 40 and the E1 protein of the papilloma virus [16].

Superfamily 4 (SF4): Helicases of this family were initially identified in bacteria and bacteriophages. SF4 helicases have a RecA-like folds core domain and contain five characteristic amino acid sequence motifs: H1, H1a, H2, H3, H4, and arginine motifs (Figure 1A). SF4 has classically studied replicative helicases such as bacterial DnaB, phage T7 gp4, phage T4 gp41, and the mitochondrial protein TWINKLE [17]. SF4 helicase domains are often closely associated with a primase domain; for example, the T7 phage gene 4 protein is a helicase-primase [16]. The gene 4 protein (gp4) of T7 bacteriophage is one of the well-studied SF4 helicases so far [13].

Superfamily 5 (SF5): Rho is the sole member of the SF5 family. It is a bacterial transcription termination factor that has RecA-like folds with Walker A, Ia, B, and R motifs. Rho translocates in a 5′ → 3′ direction along the nascent RNA transcripts to unwind RNA-DNA hybrids. It plays a role in controlling gene expression and RNA polymerase recycling.

Superfamily 6 (SF6): SF6 is comprised of archaeal and eukaryotic minichromosome maintenance (MCM) helicases. The domain of SF6 helicases exhibits an AAA+-like fold. In eukaryotes, the MCM helicase is a hexametric complex of six paralogous subunits (MCM2-7) involved in driving the progression of the replication fork [17]. Additionally, the bacterial RuvB protein is also classified within SF6. RuvB, in conjunction with RuvA and RuvC, is responsible for processing Holliday junctions during DNA repair [18].

3. DNA Metabolism at Low Temperatures

At different cellular levels, bacteria need to employ a variety of adaptation mechanisms to survive and thrive in cold environments. Key strategies include maintaining membrane fluidity, ensuring flexible protein structures, optimizing enzyme function, and mitigating the effects of low temperatures on nucleic acid structures. The elasticity of DNA is particularly temperature-dependent, with the bendability of double-stranded DNA (dsDNA) increasing as temperature rises [19]. Consequently, these temperature-induced changes in DNA’s intrinsic properties can significantly impact DNA metabolism and other related cellular processes.

3.1. DNA Replication at Subzero Temperature

Microorganisms living at low temperatures experience different mechanical properties of DNA. Low temperature is known to stabilize the secondary structures of both DNA and RNA [20]. The stabilization of DNA structures may pose difficulties for DNA replication at low temperatures. However, microorganisms have evolved and adapted to survive under extreme weather conditions. DNA polymerases are the main component of DNA replication, and their fidelity is critical for maintaining genome integrity at any given temperature. To maintain genome integrity and error-free genome duplication, faithful and proficient DNA replication at any growing temperature is crucial. DNA polymerase I of Psychromonas ingrahamii, a psychrophilic bacterium, is shown to have a higher polymerization activity than its mesophilic counterparts at a wide range of temperatures ranging from 5 °C to 50 °C [21]. Surprisingly, the DNA polymerase I of P. ingrahamii retained primer extension activity even at −19 °C [21]. Evidently, the DNA replication at subzero temperatures in Alaskan permafrost at temperatures of 0–20 °C has been demonstrated [22]. The knowledge of adaptation of life is mainly based on measurable, observable, and conceivable phenomena. Therefore, the level of our understanding of the adaptation of microorganisms to extreme environmental conditions is still primitive. However, it is comprehensible that microorganisms have evolved to faithfully replicate their genomes by circumventing any adverse effects posed by extreme environmental conditions.

3.2. DNA Topology

In E. coli cells, the negative supercoiling of plasmid DNA is increased upon cold shock. The DNA gyrase and HU (Histone-like protein) proteins of E. coli are indicated to play a role in a transient increase in the supercoiling of plasmid DNA at low temperature [23]. The negative supercoiling of DNA promotes DNA base accessibility and facilitates DNA unwinding [24]. The negative DNA supercoiling is also known to regulate DNA transcription during temperature fluctuations. Therefore, the local DNA conformational changes by negative supercoiling are believed to modulate the promoter region and influence the binding of RNA polymerase. The torsional strain generated by the negative supercoiling reduces the energy required for the strand separation and could help in DNA replication, transcription, and gene regulation processes [25,26]. Thus, it can be envisioned that low-temperature-induced stabilization of DNA structures is countered by the negative supercoiling of DNA to facilitate DNA metabolism at lower temperatures.

3.3. DNA Replication and Double-Strand Break Repair

Whether it is a psychrophile, psychrotroph, mesophile, or thermophile, each microorganism has an optimal temperature range for DNA replication. An increase or decrease in temperature beyond the optimal range can potentially affect the initiation, movement, and progression of the DNA replication fork and faithful duplication of DNA. In the psychrotolerant bacterium Pseudomonas syringae Lz4W, DNA replication endures frequent replication fork arrest and reversal at low temperature [27]. Inactivation of the recD gene, a DNA repair enzyme in P. syringae Lz4W, led to the cold sensitivity in P. syringae, and cells accumulated double-strand breaks (DSBs) at low temperature (4 °C) [28]. Later, the ATP-dependent helicase function of RecD helicase (SF1B family) was identified as essential for P. syringae’s growth at low temperature [29]. RecD is a subunit of the RecBCD enzyme [30]. In P. syringae, the RecBCD enzyme plays a crucial role in protecting cells from the low-temperature-induced DSBs [31]. In the absence of the RecBCD enzyme, P. syringae cells accumulate broken chromosomal fragments due to frequent replication fork arrest and collapse at low temperature.

The RecBCD enzyme is a heterotrimeric protein complex comprised of RecB, RecC, and RecD polypeptides. It is a highly processive DNA helicase and destructive/constructive nuclease known so far. As of our knowledge, it is also the fastest helicase observed (1000–2000 bp s⁻¹) with a greater processivity of unwinding dsDNA up to 30,000 bp per binding event under in vitro conditions [32], and >300 kb of dsDNA under in vivo conditions [33,34]. The RecBCD enzyme is a bipolar DNA helicase [35]. Interestingly, it possesses two SF1 family helicases: RecB and RecD, with opposite translocation polarities. RecB is a DNA helicase that translocates along ssDNA in the 3′ → 5′ direction and unwinds shorter duplex DNA with a 3′-overhang [36]. RecD is a DNA helicase that translocates in the 5′ → 3′ direction and unwinds shorter duplex DNA with a 5′-overhang [35]. RecB and RecD helicases therefore belong to the SF1A and SF1B families of helicase proteins, respectively. Apart from RecB and RecD helicases, RecBCD also contains another subunit known as RecC. RecC is structurally related to SF1 DNA helicase family proteins but lacks critical catalytic residues. It is apparently a defunct helicase having the characteristic helicase motifs with inactivated key residues [37]. Collectively, the RecBCD enzyme has three SF1 DNA helicases, of which two are active helicases with opposite translocation polarities (RecB and RecD) and a defunct helicase (RecC) (Figure 3).

Both RecB and RecD are poor DNA helicases on their own. They can unwind a duplex DNA only about 50 bp in size, but not beyond [29,35,36,38]. However, the unwinding rates and processivity of these subunits increase tremendously when they associate into a complex. For instance, RecB in association with RecC increases the ATP hydrolyzing ability of RecB by 27-fold on dsDNA [39]. RecB alone is unable to unwind a 105-bp DNA fragment, but it is capable of unwinding dsDNA of size more than 6000 bp when associated with RecC [38]. It suggests that RecC protein provides a structural advantage to RecB, possibly through its passive secondary translocation property [40], and contributes towards the processivity of the RecBC enzyme. The most incredible change in biochemical properties occurs when all three subunits assemble as a heterotrimeric RecBCD complex. The processivity and DNA unwinding rate increase tremendously. From the DNA unwinding ability of ~50 bp (by the individual subunits) to the staggering unwinding of 30,000 bp dsDNA per binding event [41] at the rate of 1000–2000 bp/s [42]. Therefore, as a heterotrimeric complex, RecBCD is an engineering marvel at the nanoscale. In the RecBCD crystal structure, RecB, RecC, and RecD proteins are intimately woven together in a manner by which their activities are exponentially amplified [43]. The defunct RecC helicase acts as a scaffold for both RecB and RecD motors [43]. Recently, it has been shown that the nuclease domain of RecB (the C-terminal domain of RecB) also plays a role in regulating the helicase functions of both RecB and RecD motors [44,45]. Though the RecB and RecD helicases translocate in the opposite direction (3′ → 5′ and 5′ → 3′, respectively), their architectural interaction within the complex directs them to drive in the same direction as shown in Figure 3 [46].

In P. syringae Lz4W, the RecBCD enzyme is essential for growth at low temperature. P. syringae cells lacking these subunits failed to grow and died at low temperature (4 °C) [31]. The P. syringae RecBCD enzyme is a dual helicase and nuclease. It unwinds dsDNA and degrades until it recognizes a cognate Chi-like sequence (5′-GCTGGCGC-3′) [47]. The recognition of the Chi sequence modulates the functional properties of the RecBCD enzyme and facilitates DSB repair via homologous recombination [32]. Despite P. syringae RecBCD being a highly processive helicase and nuclease, it is discovered that the helicase functions of RecD and RecB are essential for the survival of P. syringae at low temperature, not the nuclease function of the RecBCD enzyme [29,31,47]. Overall, whether it is a DNA topology, DNA synthesis, DNA replication under cold stress, or repairing broken DNA fragments, microorganisms have evolved to battle against cold environmental conditions.

4. RNA Metabolism at Low Temperatures

Similar to DNA, RNA molecules also experience temperature-dependent stabilization of secondary structures at low temperatures. The structure of RNA is relatively more sensitive to ambient temperature than DNA. The temperature-dependent structural sensitivity of RNA molecules also helps them to act as thermosensors. RNA molecules therefore play a wider role in gene regulation, RNA transcription, translation, and turnover [48,49]. The temperature-dependent stabilization of RNA secondary structures thus has a profound effect on cellular metabolism at low temperatures.

4.1. Cold Shock Response

When microorganisms experience temperature fluctuations, the most prominent response they elicit is to induce a certain group of genes that protect cells from harmful effects due to sudden changes in temperatures. The terms high, low, or normal temperatures for growth are either strain- or species-specific. Depending on the subjective high or low temperature shifts, bacteria can elicit two types of responses: heat shock response (HSR) and cold shock response (CSR). The cold shock proteins (Csps) are the most upregulated genes during CSR. Csps are small proteins ranging from 65 to 75 amino acids in length with a molecular weight of ~7 kDa. They contain a highly conserved cold shock domain (CSD) with two nucleic acid binding motifs, RNP-1 and RNP-2 [50]. Csps were first identified in E. coli [51]. About nine Csp family proteins have been identified so far, of which CspA is the highly induced protein upon cold shock. It increases at least 100-fold, reaching a level of ~10% of protein synthesis at 10 °C [50]. Interestingly, it is reported that the mRNA of cspA undergoes a temperature-dependent structural rearrangement, likely resulting in a cold-induced stabilized structure that efficiently gets translated at 10 °C, otherwise degraded at an elevated temperature (37 °C) [52]. This indicates that cspA mRNA acts like a thermosensor, modulating its protein synthesis in a temperature-dependent manner. Csps stabilize RNA structures by acting as RNA chaperones during cold adaptation [53] (Figure 4A). For detailed information on the bacterial cold shock response, refer to [54].

4.2. DEAD-Box RNA Helicases

DEAD-box proteins of RNA helicases belong to the superfamily SF2 helicases. Based on the homology with the eukaryotic initiation factor, eIF-4A, they share a consensus D-E-A-D conserved sequence in the motif-II and are the largest family by far in the SF2 family [55]. As discussed earlier, they contain a single polypeptide chain with two core RecA-like fold domains. They hydrolyze ATP upon binding to RNA and modulate RNA or RNA-protein structures [56]. In bacteria, they are generally dispensable for growth at normal temperatures, but some DEAD-box helicases are essential for growth at low temperatures [57].

E. coli possesses five DEAD-box RNA helicases: SrmB, CsdA (also known as DeaD), DbpA, RhlB, and RhlE. None of these helicases are essential for E. coli’s growth at 37 °C, but CsdA and SrmB are important for growth at low temperature (22 °C). Inactivation of csdA and srmB genes has led to a cold-sensitive phenotype. Cells lacking SrmB or CsdA were defective in 50S ribosomal assembly, indicating the importance of assistance needed for the structural rearrangement of RNA during ribosomal assembly at low temperature (22 °C) [58,59]. The helicase activity of CsDA is found essential for mRNA degradation at low temperature [60]. However, the role of these helicases at low temperature can be sometimes redundant. As an example, the RNA chaperone CspA can complement the cold-shock function of CsdA [60]. Overexpression of CsdA corrected the ribosomal defect of the srmB-deleted E. coli strain [58]. Interestingly, the functional orthologs, such as SrmB and RhlE helicases of psychrophilic Pseudoalteromonas haloplanktis and Colwellia psychrerythraea bacteria, complemented the cold-sensitive phenotypes of the rmB and csdA genes of mesophilic E. coli, respectively [57]. This reflects the functional redundancy of DEAD-box RNA helicases and their conservation across bacterial species.

The essential role of DEAD-box RNA helicases at low temperature is also observed in a foodborne psychrotrophic pathogen, Yersinia pseudotuberculosis IP32953, and in an Antarctic psychrotrophic bacterium, Pseudomonas syringae Lz4W. Both psychrotrophic bacteria contain five RNA helicases (SrmB, CsdA, DbpA, RhlB, and RhlE), similar to E. coli; however, only the CsdA helicase has been identified as essential for growth at low temperatures (3 to 4 °C) [61,62]. On the other hand, the Gram-positive bacterium Bacillus subtilis contains only four DEAD-box helicases: CshA, CshB, DeaD, and Yfml. Among the four DEAD-box helicases, the three helicases such as CshA, CshB, and Yfml are implicated in growth at low temperature [63]. Further, in a human pathogenic sporulated bacterium, Bacillus cereus ATCC 14579, among the five helicases it has (Csh A to E), CshA, CshB, and CshC were found essential for its growth at 10 °C [64]. It is perceivable that among the four to five DEAD-box RNA helicases bacteria have, one or the other DEAD-box helicases are critical for their growth at low temperatures. This further supports the idea that stabilized RNA or ribonucleoprotein (RNP) structures at low temperatures often need additional support from RNA helicases to rearrange and also assist ribonucleases in processing the stabilized RNA structures (Figure 4A).

4.3. The RNA Degradosome Complex

Degradosome is a multienzyme complex implicated in the steady-state profiles of RNA transcripts in bacteria. The RNA degradosome plays an important role in bulk mRNA degradation, processing of tRNA and 5S RNA precursors, transfer messenger RNA, and regulatory 6S RNA molecules [65]. The E. coli RNA degradosome complex consists of an endoribonuclease, RNase E; an exoribonuclease, PNPase; a RNA helicase, RhlB; the metabolic enzyme, enolase; and occasionally associating proteins such as polyphosphate kinase, DnaK, and GroEL chaperones (reviewed in [65]). In E. coli, the long C-terminal unstructured domain of RNase E acts as a scaffold for PNPase (a 3′–5′ exoribonuclease), RhlB (an ATP-dependent DEAD-box helicase), and the metabolic enzyme enolase. The DEAD-box RNA helicase RhlB helps in unwinding the secondary structures of RNA, assisting the 3′–5′ phosphorolytic exoribonuclease PNPase in degrading structured regions of RNAs [65] (Figure 4A).

The components of the degradosome complex may vary across species and under different growth conditions. In E. coli, the cold-inducible DEAD-box helicase CsdA associates with the degradosome complex at low temperatures (15 °C) [66]. It is shown that CsdA co-purifies with RNA degradosome and other components of the RNA degradosome. CsdA also exhibited the ability to replace the RhlB helicase function in the degradosome complex under in vitro conditions [66]. In the case of the aquatic Gram-negative bacterium Caulobacter crescentus, the degradosome complex includes two DEAD-box helicases, RhlB and the cold-induced RhlE, and both are essential for Caulobacter crescentus’s fitness at low temperature [67]. In the Antarctic psychrotropic bacterium P. syringe Lz4W, the degradosome complex is comprised of RNase E, RNase R, RhlE, and SrmB. Notably, the degradosome of P. syringae contains an exoribonuclease, RNase R, instead of PNPase. The P. syringae degradosome complex contains two DEAD-box helicases (RhlE and SrmB) compared to one helicase (RhlB) in the E. coli degradosome [68,69]. RNase R has dual activities; it is a helicase and exoribonuclease [70]. Although the reason behind replacing PNPase with RNase R in P. syringae is unknown, it is proposed that the helicase function of RNase R and the two DEAD-box RNA helicases are likely to play a role in the cold adaptation of P. syringae Lz4W at low temperature (4 °C), where the stabilized secondary structures of RNA are expected to occur [3,69]. The degradosome complex does not appear to play a role in cold stress response in E. coli. In P. syringae Lz4W, the degradosome complex is not essential for its growth at low temperature [71].

4.4. Ribonuclease R (RNase R)

Ribonucleases are critical for many aspects of RNA metabolism. They are essential for processing precursors, maturation, and degradation of RNA molecules. E. coli has three major processive 3′ → 5′ exoribonucleases, such as PNPase, RNase II, and RNase R [72]. PNPase is the first exoribonuclease found to interact with the E. coli degradosome [73]. It is a phosphorolytic 3′ → 5′ exoribonuclease and plays a role in the processing of 16S rRNA [72]. PNPase is essential for growth at low temperatures in E. coli and B. subtilis [74,75]. PNPase is also implicated in oxidative stress and DNA repair [76,77]. Whereas RNase II and RNase R are both hydrolytic 3′ → 5′ exoribonucleases and belong to the RNR superfamily of exoribonucleases [78]. PNPase, RNase II, and RNase R apparently have some overlapping functions in bacteria. The deletion of any of these exoribonucleases alone does not affect E. coli’s cell viability, but the combination of PNPase/RNase II and PNPase/RNase R deletions is lethal [79,80]. Among the three exoribonucleases, PNPase activity is blocked by double-stranded RNA [81], RNase II degrades only single-stranded RNA, but RNase R is the only exoribonuclease that degrades double-stranded RNA [82].

RNase R is a helicase and 3′ → 5′ exoribonuclease [70]. It efficiently degrades structured RNAs and plays an important role in many aspects of RNA metabolism [83]. RNase R and PNPase are cold-inducible proteins [84,85,86]. In E. coli, PNPase is required for growth at low temperature and DNA repair. Strangely, RNase R is essential for growth at low temperature in P. syringae Lz4W [87]. RNase R also protects P. syringae cells from oxidative stress and DNA damage [71]. P. syringae Lz4W has both RNase R and PNPase, but it is the RNase R (of P. syringae) that shows functional responsibilities similar to the PNPase of E. coli and B. subtilis. Therefore, it is suggested that P. syringae Lz4W has chosen RNase R over PNPase to support its growth at low temperature [69]. RNase R and PNPase have apparently swapped their functional responsibilities in this organism, likely for environmental needs.

Similar to E. coli RNase R, P. syringae RNase R also consists of three structural protein domains: an N-terminal cold-shock domain (CSD), a central nuclease domain (RNB domain), and a C-terminal S1 domain (S1) (Figure 4B) [71,88]. In E. coli, CSD and S1 domains of RNase R contribute towards substrate recruitment, binding, and catalysis [89]. Importantly, the RNB domain is critical for nuclease and helicase functions. The RNB domain alone can degrade the structured RNA molecules, albeit at a slower rate [89]. During the investigation of domain-dependent functions of P. syringae RNase R, it is revealed that the RNB domain alone (without the CSD and S1 domains) is sufficient to support growth at low temperature, oxidative stress, and DNA damage in P. syringae [88,90]. Interestingly, the E. coli RNase R also complemented the functions of P. syringae RNase R [89]. This indicates that RNase R, irrespective of its mesophilic or psychrophilic origin, can function and support growth at low temperatures.

Intriguingly, the exoribonuclease function of RNase R is neither required for P. syringae growth at low temperature nor protecting it from oxidative stress and DNA damage [71,88]. If the exoribonuclease function of RNase R is not required for P. syringae’s growth at low temperature, what function of RNase R other than exoribonuclease is essential for cold adaptation? The RNB domain of P. syringae RNase R is predicted to have a structure similar to the RNB domain of E. coli RNase R [71] (Figure 4B). It is proposed that the triple-helix wedge of the RNB domain of RNase R is responsible for the helicase activity similar to E. coli RNase R [91]. Since the nuclease function of RNase R is not required, it is proposed that the putative helicase function of RNase R is likely playing a role in cold adaptation and DNA repair in P. syringae Lz4W. These observations once again highlight the eminence of nucleic acid unwinding over degradation in resolving stabilized RNA structures and the survival of microorganisms at low temperatures.

5. Cold Stress: Generation of Reactive Oxygen Species

Reactive oxygen species (ROS) are produced during cellular metabolism by enzyme autoxidation. Under optimal growth conditions, hydrogen peroxide (H₂O₂), the hydroxyl radical (⋅OH), and the superoxide ion (O₂^•−) are generated at a nominal level. During stress conditions, the levels of ROS can increase, causing higher oxidative damage to biomolecules such as nucleic acids, proteins, and lipids [92,93]. Hence, organisms have developed antioxidant defense systems (ADS) to protect themselves from oxidative damage [94]. The ADS includes enzymes: superoxide dismutase, catalase, glutathione peroxidase, and reductase, and antioxidant substances: glutathione and ascorbic acid. Superoxide dismutase converts O₂^•− to H₂O₂, and catalase decomposes H₂O₂ to water molecules.

The cold shock and oxidative stress responses are interconnected. In E. coli, the cold shock response elevated the intracellular level of superoxide anion, causing an increase in ROS at low temperatures similar to oxidative stress [95]. In the Antarctic bacterium Pseudomonas fluorescens MTCC 667, growth at low temperature (4 °C) increased the production of free radicals and also the activities of oxygen-scavenging superoxide dismutase and catalase enzymes at 4 °C compared to 22 °C [96]. Further in P. syringae Lz4W, cells lacking DNA repair RecBCD and RNA-degrading exoribonuclease RNase R enzymes were sensitive to oxidative stress and DNA-damaging agents [27,31,71]. Interestingly, their nuclease activity is not required; instead, the helicase function of both enzymes is essential for protecting cells from oxidative stress and DNA damage [27,31,71]. Low temperature is also known to induce intracellular oxidative stress in the phytopathogen bacterium Pseudomonas savastanoi pv. phaseolicola NPS3121 [97]. A correlation between low temperature and oxidative stress has been observed in Saccharomyces cerevisiae, where research indicates that low temperature growth is closely associated with increased oxidative stress [98].

Cold stress can also have a devastating effect on plants. Cold stress is shown to increase the level and activity of various ROS-scavenging enzymes in plants (reviewed in [99]). Low temperature stress is demonstrated to induce H₂O₂ production in plants [100]. Plants can sense the low temperature stress through changes in membrane fluidity, alterations in plant hormones, the production and accumulation of ROS, calcium, nitric oxide signaling, and other stress response cascades [101]. ROS induces calcium signaling and influences cold regulation of gene expression. Low temperature stress has been shown to increase ROS, antioxidants, and osmatic adjustment substances in rice (Oryza sativa L.), particularly at the reproductive stage, affecting rice production [102]. OsBIRH1, a DEAD-box helicase, is known to modulate the defense responses against infection and oxidative stress in rice [103]. How does low temperature induce oxidative stress? Oxygen solubility is known to increase with a decrease in temperature in water and other electrolytes [104], indicating that increased oxygen solubility could elevate ROS production, leading to higher oxidative stress at low temperatures.

6. Role of Helicases in Cold Adaptation of Plants

RNA helicases, particularly those of the SF2 superfamily, play a critical role in the adaptation/acclimatization of plants to low temperatures. Here, we emphasize RNA helicases and their importance in the cold adaptation of plants. Low temperatures can have two types of stresses on plants: chilling stress (0–20 °C) and freezing stress (<0 °C). Both stresses can cause devastating effects on plants’ growth, development, and crop production. Many major crops, including maize (Zea mays), rice (Oryza sativa), soybean (Glycine max), tomato (Solanum lycopersicum), and cotton (Gossypium hirsutum), are not acclimatized to cold conditions. However, recent global temperature fluctuations could also make these major crops endure temperature-dependent stress. In plants, the low temperature stabilized secondary structures of RNA can also adversely affect the processes of RNA splicing, RNA transcript turnover, transcription, and RNA decay at low temperatures. Similar to bacteria, many plants have also evolved to withstand the cold stress through various cellular mechanisms.

More than hundreds of DEAD-box, DEAH-box, Ski2-like, and Swif2/Snf2 family RNA helicase genes of the SF2 superfamily have been identified in plants [105]. In Arabidopsis thaliana alone, more than 177 genes encoding RNA helicases have been annotated, of which 56 are the DEAD-box helicase-coding genes [106]. In rice, about 200 RNA helicase-encoding genes have also been annotated [106]. DEAD box helicases such as AtRH3, AtRH7, AtRH9, AtRH25, AtRH27, AtHELPS, RCF-1, and LOS4-2 play a vital role during cold stress in Arabidopsis. These helicases have been shown to play essential roles in rRNA biogenesis, pre-mRNA splicing, mRNA transport, and ribosomal formation under stress (reviewed in [107]) (Figure 5). They enhance freezing tolerance, growth, and development of Arabidopsis at low temperatures [108,109,110]. In rice (Oryza sativa), the OsABP, OsRH17, OsRH42, and TCD33 RNA helicases are also reported to play important roles in cold stress [107]. RNA helicase 42 (OsRH42) in pre-mRNA splicing [111]; OsBIRH1, a chloroplast-localized DEAD-box helicase in chloroplast function; and TCD33 helicase for chloroplast development at the seedling stage found critical under cold stress in rice [112,113,114] (Figure 5). DEAD-box helicases such as the GmRH helicase of soybean and TaRH1 of wheat are also indicated to be cold stress [115,116].

7. Significance of Nucleic Acids Unwinding over Degradation in Survival of Microorganisms at Low Temperatures

Some helicases possess both helicase and nuclease functions. The DNA repair enzymes such as RecBCD, AddAB, and AdnAB have both helicase and nuclease functions [117]. The RecBCD enzyme is a very robust helicase and a highly destructive nuclease. As discussed earlier, RecBCD can unwind and degrade duplex DNA of size > 300 kb at the rate of 1000–2000 bp/s [33,34,118]. The nuclease activity of RecBCD is crucial for degrading the linearized chromosomal DNA. RecBCD can degrade linearized chromosomal DNA of size 300 kb in vivo [34]. RecBCD can even degrade the entire linearized E. coli chromosome within several hours from the broken end [119]. Despite RecBCD having such an impressive nuclease function, its nuclease function is surprisingly dispensable for the growth of P. syringae at low temperature [31,47]. Intriguingly, the helicase function of the RecBCD enzyme is crucial, not the nuclease activity. How does DNA unwinding play a crucial role during growth at low temperatures? Again, it is possible that the stabilization of secondary DNA structures at low temperatures arguably makes helicase function more consequentially important than the nuclease function. Since multiple exonucleases are redundantly involved in nucleolytic degradation, DNA repair, and homologous recombination processes [120], the nuclease activity of RecBCD is possibly of less importance than its unwinding function. The information provided in this section therefore clearly indicates that the DNA unwinding function is critical under cold stress.

In the case of RNA helicases, RNase R, a 3′ → 5′ exoribonuclease, has both nuclease and helicase activities [70]. Hence, it is capable of processing highly structured RNA substrates [82] and is essential for growth at low temperature in P. syringae Lz4W [87]. In the process of understanding the functional role of RNase R in cold adaptation in P. syringae, it was discovered that the putative helicase function of RNase R is critical for cold adaptation and oxidative stress in P. syringae, not the exoribonuclease function [69,71,88]. This further corroborates the significance of nucleic acids unwinding over the degradation function in cold adaptation, stress, and acclimatization.

Based on the literature discussed in this article, it is comprehensible that helicases play an indisputable role in life adaptation at low temperatures. The challenges posed by low temperatures on the mechanical, structural, and functional properties of nucleic acids, proteins, and cellular membranes require machineries that can circumvent the adverse situations and help life to thrive at low temperatures. In this perspective, the role of helicases in cold adaptation, cold stress, and acclimatization is irrefutable.