Table of Contents
- Introduction
- Structure of DNA helicases
- Types of DNA helicases
- Mechanism of DNA helicases
- Functions of DNA helicases
- Examples of DNA helicases
Introduction
- DNA helicases are essential enzymes present across all domains of life, playing a crucial role in various nucleic acid metabolic processes, including DNA replication, transcription, translation, DNA repair, recombination, ribosome biogenesis, and decay.
- DNA helicases were first discovered in E. coli in 1976.
- These enzymes are ATP (adenosine triphosphate)-dependent and facilitate the separation of the two parental DNA strands by unwinding the DNA at a specific position known as the origin of replication, where replication begins.
- The unwinding creates a replication fork, which moves progressively away from the origin as the DNA continues to unwind.
- The unwinding of the DNA helix at the replication fork is primarily catalyzed by two DNA helicases working together: one helicase operates on the leading strand, and the other on the lagging strand.
- DNA helicases utilize ATP as an energy source to disrupt the hydrogen bonds between the nitrogenous base pairs of double-stranded DNA.
- In addition to unwinding double-stranded DNA, some DNA helicases also:
- Unwind DNA triplexes or G-quadruplexes.
- Displace proteins bound to single- or double-stranded DNA.
- All DNA helicases share three fundamental biochemical properties:
- Nucleic acid binding.
- NTP/dNTP binding and hydrolysis.
- NTP/dNTP hydrolysis-dependent unwinding of duplex nucleic acids in either the 3’ to 5’ or 5’ to 3’ direction.
Structure of DNA helicases
- DNA helicases can be categorized into two structural types: those that form hexameric ring structures and those that do not.
- Helicases with a ring structure feature a central channel that encircles the nucleic acid. The stability and processivity of these enzymes increase as the topological interactions between the protein and the nucleic acid become more extensive.
- Most hexameric helicases are homohexamers, except for the eukaryotic minichromosomal maintenance (MCM) helicases.
- Helicase motifs refer to nine conserved short amino acid sequence fingerprints, labeled Q, I, Ia, Ib, II, III, IV, V, and VI, found in the helicases of various organisms.
- These conserved motifs are typically clustered within the central region of the helicase, spanning 200 to 700 amino acids.
Types of DNA helicases
- Numerous DNA helicases have been isolated from single cells to accommodate the diverse structural requirements of substrates during various stages of DNA transactions. For example:
- E. coli: 14 DNA helicases
- Viruses: 12 DNA helicases
- Bacteriophages: 6 DNA helicases
- Plants: 8 DNA helicases
- Yeast: 15 DNA helicases
- Calf thymus: 11 DNA helicases
- Human cells: Approximately 24 DNA helicases
- DNA helicases are classified into six superfamilies (SF1 to SF6) based on conserved helicase motifs.
- Toroidal (ring-forming) hexameric helicases belong to SF3 to SF6, while non-ring-forming helicases are categorized under SF1 and SF2.
- Ring-forming helicases encircle DNA and translocate in a processive manner.
Superfamily SF1:
- SF1 helicases have a common core structure comprising two α-β RecA-like domains and function as monomers involved in recombination, transcription, repair, and other processes.
- Their structural similarity to the RecA recombination protein includes tandem alpha-helices and five parallel beta-strands.
- ATP binds to the amino-proximal α-β domain, containing motif I (Walker A) and motif II (Walker B).
- Motif III (S-A-T) in the N-terminal domain links ATPase activity to helicase function.
- The carboxy-terminal α-β domain resembles the proximal domain but lacks an ATP-binding site, likely due to gene duplication.
- SF1 is divided into three subfamilies:
- PiF1/RecD
- Rep/UvrD
- UpF1-like
- Translocation direction on single-stranded DNA:
- SF1A: 3' to 5'
- SF1B: 5' to 3'
- Crystal structures include:
- SF1A helicases: PcrA, UvrD
- SF1B helicases: RecD2, Dda
Crystal structures of SF1A (PcrA, UvrD) and SF1B (RecD2, Dda) helicases. Image Source: Raney, K. D., Byrd, A. K., & Aarattuthodiyil, S. (2013). Structure and Mechanisms of SF1 DNA Helicases. Advances in experimental medicine and biology, 767, 17–46. https://doi.org/10.1007/978-1-4614-5037-5_2
Superfamily SF2:
- SF2 helicases play a critical role in RNA metabolism and various processes in DNA metabolism.
- SF2 includes 10 separate families, each with a distinct role in nucleic acid metabolism.
Superfamily SF3:
- SF3 helicases are predominantly encoded by small DNA/RNA viruses and large nucleocytoplasmic DNA viruses.
- These helicases feature a spacer region between Walker A and Walker B motifs, with a third motif (C) located between Walker B and the conserved C-terminal region.
Superfamily SF4:
- SF4 helicases are hexameric and function primarily in bacterial or bacteriophage replication (e.g., bacterial DnaB protein).
- The central core structure resembles the α-β RecA-like domain.
Superfamily SF5:
- SF5 helicases include the E. coli Rho factor, a hexamer that:
- Terminates specific RNA transcripts.
- Translocates exclusively on RNA.
- Provides insights into the mechanisms of replicative helicases.
Superfamily SF6:
- SF6 helicases feature an AAA+ core, absent in SF3 helicases.
- Examples include minichromosome maintenance (MCM) helicases and related proteins such as RuvA, RuvB, and RuvC.
Mechanism of DNA helicases
Figure: Proposed Brownian ratchet mechanism and most likely kinetic scheme. Image Source: Burnham, D.R., Kose, H.B., Hoyle, R.B. et al. The mechanism of DNA unwinding by the eukaryotic replicative helicase. Nat Commun 10, 2159 (2019). https://doi.org/10.1038/s41467-019-09896-2
- DNA helicases are vital motor proteins that unwind duplex DNA to produce transient single-stranded DNA intermediates required for replication, recombination, and repair.
- While helicases share a similar three-dimensional fold, they assemble into various oligomeric states to achieve full activity, with the hexameric assembly being the most established.
- In the hexameric helicase class, six subunits form a ring-shaped structure for efficient function.
- Within the double helix, the oligomerization of subunits is stabilized by the binding of nucleoside triphosphates (NTPs) or metal ions.
- Of the six potential ATP-binding sites in the hexamer:
- Two subunits bind ATP tightly.
- Two subunits bind ADP and Pi.
- Two subunits remain empty.
- The proximity between ATP molecules and ATP-binding sites is critical for forming covalent bonds with the enzyme and the sugar-phosphate backbone of DNA.
- The energy released from ATP hydrolysis helps overcome the activation barrier necessary for DNA strand separation.
- During ATP hydrolysis, the three states (ATP-bound, ADP-bound, and empty) interconvert in a coordinated manner, creating a ripple effect.
- The ripple effect triggers conformational changes in the helicase ring, causing a loop to extend into the central channel of the ring and bind to the DNA.
- The oscillating motion of the loop pulls the DNA strand through the central hole, separating the DNA double helix into single strands.
Functions of DNA helicases
- DNA helicases unwind or separate the hydrogen bonds between nucleotide bases of two strands of double-stranded DNA using energy derived from ATP.
- DNA helicases are essential for maintaining homologous somatic genome stability and for meiotic mixing of parental genomes in plants.
- FANCJ is a DNA helicase associated with ovarian cancer, hereditary breast cancer, and Fanconi anemia (a progressive bone marrow failure disorder). Mutations in FANCJ disrupt its helicase activity.
- FANCJ is involved in cancer suppression and directly interacts with BRCA1 for double-strand break repair.
- Certain helicases, such as RECQL1, RECQL4, RECQL5, WRN, and BLM, are involved in strand annealing, promoting base pairing between strands.
- DNA helicases play an active role in the transmission of genetic information from one generation to the next.
Examples of DNA helicases
E. coli DNA helicases:
- DnaB: A replicative helicase that operates at the replication fork, primarily on the lagging strand.
- Helicase II (UvrD): Involved in nucleotide excision repair.
Bacteriophages DNA helicases:
T4 UvsW: Catalyzes branch migration and plays a crucial role in DNA recombination, regulation of origin of replication, and DNA repair.
Viral DNA helicases:
HSV-1 UL9 protein: An origin binding protein essential for the initiation of HSV replication.
Yeast DNA helicases:
PIF1: Functions in mitochondrial DNA repair and recombination.
Human DNA helicases:
HDH VI: Prefers substrate structures similar to those at the replication fork.
References
- Alexander Knoll, Holger Puchta. (2011). The role of DNA helicases and their interaction partners in genome stability and meiotic recombination in plants. Journal of Experimental Botany, 62(5), 1565–1579. https://doi.org/10.1093/jxb/erq357
- Burnham, D.R., Kose, H.B., Hoyle, R.B. et al. (2019). The mechanism of DNA unwinding by the eukaryotic replicative helicase. Nat Commun, 10, 2159. https://doi.org/10.1038/s41467-019-09896-2
- Fairman-Williams, M. E., Guenther, U. P., & Jankowsky, E. (2010). SF1 and SF2 helicases: family matters. Current opinion in structural biology, 20(3), 313-324.
- Lewin, B. (2007). Genes IX. Oxford University Press, and Cell Press.
- Patel, S.S., Donmez, I. (2006). Mechanisms of helicases. J Biol Chem, 281(27), 18265-8. doi: 10.1074/jbc.R600008200. Epub 2006 May 2. PMID: 16670085.
- Raney, K.D., Byrd, A.K., Aarattuthodiyil, S. (2013). Structure and Mechanisms of SF1 DNA Helicases. Adv Exp Med Biol, 767, 17-46. doi:10.1007/978-1-4614-5037-5_2
- Tuteja, N., & Tuteja, R. (2004). Unraveling DNA helicases: motif, structure, mechanism, and function. European Journal of Biochemistry, 271(10), 1849-1863.
- Tuteja, N., & Tuteja, R. (2004). Prokaryotic and eukaryotic DNA helicases: essential molecular motor proteins for cellular machinery. European Journal of Biochemistry, 271(10), 1835-1848.
- Uchiumi, F., Seki, M., Furuichi, Y., eds. (2015). DNA Helicases: Expression, Functions and Clinical Implications. Lausanne: Frontiers Media. doi: 10.3389/978-2-88919-575-6
- Verma, P. S., & Agrawal, V. K. (2006). Cell Biology, Genetics, Molecular Biology, Evolution & Ecology (1st ed.). S. Chand and Company Ltd.
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