Prime DNA Synthesis Research Articles

How Pol α-primase is targeted to replisomes to prime eukaryotic DNA replication

During eukaryotic DNA replication, Pol α-primase generates primers at replication origins to start leading-strand synthesis and every few hundred nucleotides during discontinuous lagging-strand replication. How Pol α-primase is targeted to replication forks to prime DNA synthesis is not fully understood. Here, by determining cryoelectron microscopy (cryo-EM) structures of budding yeast and human replisomes containing Pol α-primase, we reveal a conserved mechanism for the coordination of priming by the replisome. Pol α-primase binds directly to the leading edge of the CMG (CDC45-MCM-GINS) replicative helicase via a complex interaction network. The non-catalytic PRIM2/Pri2 subunit forms two interfaces with CMG that are critical for invitro DNA replication and yeast cell growth. These interactions position the primase catalytic subunit PRIM1/Pri1 directly above the exit channel for lagging-strand template single-stranded DNA (ssDNA), revealing why priming occurs efficiently only on the lagging-strand template and elucidating a mechanism for Pol α-primase to overcome competition from RPA to initiate primer synthesis.

Reverse transcriptases prime DNA synthesis.

The discovery of reverse transcriptases (RTs) challenged the central dogma by establishing that genetic information can also flow from RNA to DNA. Although they act as DNA polymerases, RTs are distantly related to replicases that also possess de novo primase activity. Here we identify that CRISPR associated RTs (CARTs) directly prime DNA synthesis on both RNA and DNA. We demonstrate that RT-dependent priming is utilized by some CRISPR-Cas complexes to synthesise new spacers and integrate these into CRISPR arrays. Expanding our analyses, we show that primer synthesis activity is conserved in representatives of other major RT classes, including group II intron RT, telomerase and retroviruses. Together, these findings establish a conserved innate ability of RTs to catalyse de novo DNA primer synthesis, independently of accessory domains or alternative priming mechanisms, which likely plays important roles in a wide variety of biological pathways.

Predicted structure of the hepatitis B virus polymerase reveals an ancient conserved protein fold.

Hepatitis B virus (HBV) chronically infects >250 million people. It replicates by a unique protein-primed reverse transcription mechanism, and the primary anti-HBV drugs are nucleos(t)ide analogs targeting the viral polymerase (P). P has four domains compared to only two in most reverse transcriptases: the terminal protein (TP) that primes DNA synthesis, a spacer, the reverse transcriptase (RT), and the ribonuclease H (RNase H). Despite being a major drug target and catalyzing a reverse transcription pathway very different from the retroviruses, HBV P has resisted structural analysis for decades. Here, we exploited computational advances to model P. The TP wrapped around the RT domain rather than forming the anticipated globular domain, with the priming tyrosine poised over the RT active site. The orientation of the RT and RNase H domains resembled that of the retroviral enzymes despite the lack of sequences analogous to the retroviral linker region. The model was validated by mapping residues with known surface exposures, docking nucleic acids, mechanistically interpreting mutations with strong phenotypes, and docking inhibitors into the RT and RNase H active sites. The HBV P fold, including the orientation of the TP domain, was conserved among hepadnaviruses infecting rodent to fish hosts and a nackednavirus, but not in other non-retroviral RTs. Therefore, this protein fold has persisted since the hepadnaviruses diverged from nackednaviruses >400 million years ago. This model will advance mechanistic analyses into the poorly understood enzymology of HBV reverse transcription and will enable drug development against non-active site targets for the first time.

A Role for Human DNA Polymerase λ in Alternative Lengthening of Telomeres.

Telomerase negative cancer cell types use the Alternative Lengthening of Telomeres (ALT) pathway to elongate telomeres ends. Here, we show that silencing human DNA polymerase (Pol λ) in ALT cells represses ALT activity and induces telomeric stress. In addition, replication stress in the absence of Pol λ, strongly affects the survival of ALT cells. In vitro, Pol λ can promote annealing of even a single G-rich telomeric repeat to its complementary strand and use it to prime DNA synthesis. The noncoding telomeric repeat containing RNA TERRA and replication protein A negatively regulate this activity, while the Protection of Telomeres protein 1 (POT1)/TPP1 heterodimer stimulates Pol λ. Pol λ associates with telomeres and colocalizes with TPP1 in cells. In summary, our data suggest a role of Pol λ in the maintenance of telomeres by the ALT mechanism.

A novel fluorescent aptasensor for aflatoxin M1 detection using rolling circle amplification and g-C3N4 as fluorescence quencher

Aflatoxin M1 is a prevailing natural carcinogenic mycotoxin, found in milk. Herein, we reported a sensitive fluorescent aptasensor for AFM1, based on rolling circle amplification (RCA) technology to improve the sensitivity of the assay. The detection of AFM1 was achieved using KYF4: Eu3+ time-resolved fluorescent nanoparticles (KYF4: Eu3+ TRFNPs) as signal probe and graphitic carbon nitride (g-C3N4) nanosheet as a quencher. The AFM1 aptamer act as a recognition element and as a primer for DNA synthesis. TRFNPs attached to complementary DNA (TRFNPs-cDNA) acts as a signal probe. In the absence of target, a rolling circle template (RCT) ligated to aptamer and RCA reaction initiated. The TRFNPs-cDNA was strengthened to amplified RCA product (RCAP) to make RCAP/TRFNPs-cDNA duplex. This duplex cannot be adsorbed onto g-C3N4, and ultimately no fluorescence quenched. In the presence of AFM1, aptamer recognized the target and aptamer-target complex was formed. Hence, no aptamer hybridized with RCT, therefore, no RCA and duplex formation. Consequently, free TRFNPs-cDNA adsorbed onto g-C3N4 and resulted in quenched fluorescence. The assay provides a lower detection limit (0.0194 pg/mL) than the previously reported assays and has good recoveries between 92–99.8 %. Hence, the present method would be a suitable method for the quantification of small molecules.

SMART (Single Molecule Analysis of Resection Tracks) Technique for Assessing DNA end-Resection in Response to DNA Damage.

DNA double strand breaks (DSBs) are among the most toxic lesions affecting genome integrity. DSBs are mainly repaired through non-homologous end joining (NHEJ) and homologous recombination (HR). A crucial step of the HR process is the generation, through DNA end-resection, of a long 3' single-strand DNA stretch, necessary to prime DNA synthesis using a homologous region as a template, following DNA strand invasion. DNA end resection inhibits NHEJ and triggers homology-directed DSB repair, ultimately guaranteeing a faithful DNA repair. Established methods to evaluate the DNA end-resection process are the immunofluorescence analysis of the phospho-S4/8 RPA32 protein foci, a marker of DNA end-resection, or of the phospho-S4/8 RPA32 protein levels by Western blot. Recently, the Single Molecule Analysis of Resection Tracks (SMART) has been described as a reliable method to visualize, by immunofluorescence, the long 3' single-strand DNA tails generated upon cell treatment with a S-phase specific DNA damaging agent (such as camptothecin). Then, DNA tract lengths can be measured through an image analysis software (such as Photoshop), to evaluate the processivity of the DNA end-resection machinery. The preparation of DNA fibres is performed in non-denaturing conditions so that the immunofluorescence detects only the specific long 3' single-strand DNA tails, generated from DSB processing.

Initiation of homologous recombination at DNA nicks

Discontinuities in only a single strand of the DNA duplex occur frequently, as a result of DNA damage or as intermediates in essential nuclear processes and DNA repair. Nicks are the simplest of these lesions: they carry clean ends bearing 3′-hydroxyl groups that can undergo ligation or prime new DNA synthesis. In contrast, single-strand breaks also interrupt only one DNA strand, but they carry damaged ends that require clean-up before subsequent steps in repair. Despite their apparent simplicity, nicks can have significant consequences for genome stability. The availability of enzymes that can introduce a nick almost anywhere in a large genome now makes it possible to systematically analyze repair of nicks. Recent experiments demonstrate that nicks can initiate recombination via pathways distinct from those active at double-strand breaks (DSBs). Recombination at targeted DNA nicks can be very efficient, and because nicks are intrinsically less mutagenic than DSBs, nick-initiated gene correction is useful for genome engineering and gene therapy. This review revisits some physiological examples of recombination at nicks, and outlines experiments that have demonstrated that nicks initiate homology-directed repair by distinctive pathways, emphasizing research that has contributed to our current mechanistic understanding of recombination at nicks in mammalian cells.

In Vitro Mutagenesis Using Double-Stranded DNA Templates: Selection of Mutants with DpnI

In this protocol, two oligonucleotides are used to prime DNA synthesis by a high-fidelity polymerase on a denatured plasmid template. The two oligonucleotides both contain the desired mutation and have the same starting and ending positions on opposite strands of the plasmid DNA. The entire lengths of both strands of the plasmid DNA are amplified in a linear fashion during several rounds of thermal cycling, generating a mutated plasmid containing staggered nicks on opposite strands. Because of the amount of template DNA used in the amplification reaction, the background of transformed colonies containing wild-type plasmid DNA can be quite high unless steps are taken to enrich for mutant molecules. In this protocol, the products of the linear amplification reaction are treated with the restriction enzyme DpnI, which specifically cleaves fully methylated GMe6ATC sequences. DpnI will therefore digest the bacterially generated DNA used as template for amplification, but it will not digest DNA synthesized during the course of the reaction in vitro. DpnI-resistant molecules, which are rich in the desired mutants, are recovered by transforming E. coli cells to antibiotic resistance. Because the method works well with virtually any plasmid of moderate size (<7 kb), it can be used to introduce mutations directly into full-length cDNAs and eliminates the need for subcloning into specialized vectors.

The Yeast Mitochondrial RNA Polymerase and Transcription Factor Complex Catalyzes Efficient Priming of DNA Synthesis on Single-stranded DNA

Primases use single-stranded (ss) DNAs as templates to synthesize short oligoribonucleotide primers that initiate lagging strand DNA synthesis or reprime DNA synthesis after replication fork collapse, but the origin of this activity in the mitochondria remains unclear. Herein, we show that the Saccharomyces cerevisiae mitochondrial RNA polymerase (Rpo41) and its transcription factor (Mtf1) is an efficient primase that initiates DNA synthesis on ssDNA coated with the yeast mitochondrial ssDNA-binding protein, Rim1. Both Rpo41 and Rpo41-Mtf1 can synthesize short and long RNAs on ssDNA template and prime DNA synthesis by the yeast mitochondrial DNA polymerase Mip1. However, the ssDNA-binding protein Rim1 severely inhibits the RNA synthesis activity of Rpo41, but not the Rpo41-Mtf1 complex, which continues to prime DNA synthesis efficiently in the presence of Rim1. We show that RNAs as short as 10-12 nt serve as primers for DNA synthesis. Characterization of the RNA-DNA products shows that Rpo41 and Rpo41-Mtf1 have slightly different priming specificity. However, both prefer to initiate with ATP from short priming sequences such as 3'-TCC, TTC, and TTT, and the consensus sequence is 3'-Pu(Py)2-3 Based on our studies, we propose that Rpo41-Mtf1 is an attractive candidate for serving as the primase to initiate lagging strand DNA synthesis during normal replication and/or to restart stalled replication from downstream ssDNA.

Protein-Primed Replication of Bacteriophage Φ29 DNA.

The requirement of DNA polymerases for a 3'-hydroxyl (3'-OH) group to prime DNA synthesis raised the question about how the ends of linear chromosomes could be replicated. Among the strategies that have evolved to handle the end replication problem, a group of linear phages and eukaryotic and archaeal viruses, among others, make use of a protein (terminal protein, TP) that primes DNA synthesis from the end of their genomes. The replicative DNA polymerase recognizes the OH group of a specific residue in the TP to initiate replication that is guided by an internal 3' nucleotide of the template strand. By a sliding-back mechanism or variants of it the terminal nucleotide(s) is(are) recovered and the TP becomes covalently attached to the genome ends. Bacillus subtilis phage ϕ29 is the organism in which such a mechanism has been studied more extensively, having allowed to lay the foundations of the so-called protein-primed replication mechanism. Here we focus on the main biochemical and structural features of the two main proteins responsible for the protein-primed initiation step: the DNA polymerase and the TP. Thus, we will discuss the structural determinants of the DNA polymerase responsible for its ability to use sequentially a TP and a DNA as primers, as well as for its inherent capacity to couple high processive synthesis to strand displacement. On the other hand, we will review how TP primes initiation followed by a transition step for further DNA-primed replication by the same polymerase molecule. Finally, we will review how replication is compartmentalized in vivo.

The C-terminal Domain of the DNA Polymerase Catalytic Subunit Regulates the Primase and Polymerase Activities of the Human DNA Polymerase α-Primase Complex

The initiation of DNA synthesis during replication of the human genome is accomplished primarily by the DNA polymerase α-primase complex, which makes the RNA-DNA primers accessible to processive DNA pols. The structural information needed to understand the mechanism of regulation of this complex biochemical reaction is incomplete. The presence of two enzymes in one complex poses the question of how these two enzymes cooperate during priming of DNA synthesis. Yeast two-hybrid and direct pulldown assays revealed that the N-terminal domain of the large subunit of primase (p58N) directly interacts with the C-terminal domain of the catalytic subunit of polα (p180C). We found that a complex of the C-terminal domain of the catalytic subunit of polα with the second subunit (p180C-p70) stimulated primase activity, whereas the whole catalytically active heterodimer of polα (p180ΔN-p70) inhibited RNA synthesis by primase. Conversely, the polα catalytic domain without the C-terminal part (p180ΔN-core) possessed a much higher propensity to extend the RNA primer than the two-subunit polα (p180ΔN-p70), suggesting that p180C and/or p70 are involved in the negative regulation of DNA pol activity. We conclude that the interaction between p180C, p70, and p58 regulates the proper primase and polymerase function. The composition of the template DNA is another important factor determining the activity of the complex. We have found that polα activity strongly depends on the sequence of the template and that homopyrimidine runs create a strong barrier for DNA synthesis by polα.

Identification of RNA binding motifs in the R2 retrotransposon-encoded reverse transcriptase.

R2 non-LTR retrotransposons insert at a specific site in the 28S rRNA genes of many animal phyla. R2 elements encode a single polypeptide with reverse transcriptase, endonuclease and nucleic acid binding domains. Integration involves separate cleavage of the two DNA strands at the target site and utilization of the released 3′ ends to prime DNA synthesis. Critical to this integration is the ability of the protein to specifically bind 3′ and 5′ regions of the R2 RNA. In this report, alanine mutations in two conserved motifs N-terminal to the reverse transcriptase domain were generated and shown to result in proteins that retained the ability to cleave the first strand of the DNA target, to reverse transcribe RNA from an annealed primer and to displace annealed RNA when using DNA as a template. However, the mutant proteins had greatly reduced ability to bind 3′ and 5′ RNA in mobility shift assays, use the DNA target to prime reverse transcription and conduct second-strand DNA cleavage. These motifs thus appear to participate in all activities of the R2 protein known to require specific RNA binding. The similarity of these R2 RNA binding motifs to those of telomerase and group II introns is discussed.

Polymerase theta-mediated end joining of replication-associated DNA breaks in C. elegans

DNA lesions that block replication fork progression are drivers of cancer-associated genome alterations, but the error-prone DNA repair mechanisms acting on collapsed replication are incompletely understood, and their contribution to genome evolution largely unexplored. Here, through whole-genome sequencing of animal populations that were clonally propagated for more than 50 generations, we identify a distinct class of deletions that spontaneously accumulate in C. elegans strains lacking translesion synthesis (TLS) polymerases. Emerging DNA double-strand breaks are repaired via an error-prone mechanism in which the outermost nucleotide of one end serves to prime DNA synthesis on the other end. This pathway critically depends on the A-family polymerase theta, which protects the genome against gross chromosomal rearrangements. By comparing the genomes of isolates of C. elegans from different geographical regions, we found that in fact most spontaneously evolving structural variations match the signature of polymerase theta-mediated end joining (TMEJ), illustrating that this pathway is an important source of genetic diversification.

A highly divergent archaeo-eukaryotic primase from the Thermococcus nautilus plasmid, pTN2

We report the characterization of a DNA primase/polymerase protein (PolpTN2) encoded by the pTN2 plasmid from Thermococcus nautilus. Sequence analysis revealed that this protein corresponds to a fusion between an N-terminal domain homologous to the small catalytic subunit PriS of heterodimeric archaeal and eukaryotic primases (AEP) and a C-terminal domain related to their large regulatory subunit PriL. This unique domain configuration is not found in other virus- and plasmid-encoded primases in which PriS-like domains are typically fused to different types of helicases. PolpTN2 exhibited primase, polymerase and nucleotidyl transferase activities and specifically incorporates dNTPs, to the exclusion of rNTPs. PolpTN2 could efficiently prime DNA synthesis by the T. nautilus PolB DNA polymerase, suggesting that it is used in vivo as a primase for pTN2 plasmid replication. The N-terminal PriS-like domain of PolpTN2 exhibited all activities of the full-length enzyme but was much less efficient in priming cellular DNA polymerases. Surprisingly, the N-terminal domain possesses reverse transcriptase activity. We speculate that this activity could reflect an ancestral function of AEP proteins in the transition from the RNA to the DNA world.

Evolution of the R2 Retrotransposon Ribozyme and Its Self-Cleavage Site

R2 is a non-long terminal repeat retrotransposon that inserts site-specifically in the tandem 28S rRNA genes of many animals. Previously, R2 RNA from various species of Drosophila was shown to self-cleave from the 28S rRNA/R2 co-transcript by a hepatitis D virus (HDV)-like ribozyme encoded at its 5' end. RNA cleavage was at the precise 5' junction of the element with the 28S gene. Here we report that RNAs encompassing the 5' ends of R2 elements from throughout its species range fold into HDV-like ribozymes. In vitro assays of RNA self-cleavage conducted in many R2 lineages confirmed activity. For many R2s, RNA self-cleavage was not at the 5' end of the element but at 28S rRNA sequences up to 36 nucleotides upstream of the junction. The location of cleavage correlated well with the types of endogenous R2 5' junctions from different species. R2 5' junctions were uniform for most R2s in which RNA cleavage was upstream in the rRNA sequences. The 28S sequences remaining on the first DNA strand synthesized during retrotransposition are postulated to anneal to the target site and uniformly prime second strand DNA synthesis. In species where RNA cleavage occurred at the R2 5' end, the 5' junctions were variable. This junction variation is postulated to result from the priming of second strand DNA synthesis by chance microhomologies between the target site and the first DNA strand. Finally, features of R2 ribozyme evolution, especially changes in cleavage site and convergence on the same active site sequences, are discussed.

AXM mutagenesis: An efficient means for the production of libraries for directed evolution of proteins

Affinity maturation is an important part of the recombinant antibody development process. There are several well-established approaches for generating libraries of mutated antibody genes for affinity maturation, but these approaches are generally too laborious or expensive to allow high-throughput, parallel processing of multiple antibodies. Here, we describe a scalable approach that enables the generation of libraries with greater than 108 clones from a single Escherichia coli transformation. In our method, a mutated DNA fragment is produced using PCR conditions that promote nucleotide misincorporation into newly synthesized DNA. In the PCR reaction, one of the primers contains at least three phosphorothioate linkages at its 5′ end, and treatment of the PCR product with a 5′ to 3′ exonuclease is used to preferentially remove the strand synthesized with the non-modified primer, resulting in a single-stranded DNA fragment. This fragment then serves as a megaprimer to prime DNA synthesis on a uracilated, circular, single-stranded template in a Kunkel-like mutagenesis reaction that biases nucleotide base-changes between the megaprimer and uracilated DNA sequence in favor of the in vitro synthesized megaprimer. This method eliminates the inefficient subcloning steps that are normally required for the construction of affinity maturation libraries from randomly mutagenized antibody genes.

Mechanism for priming DNA synthesis by yeast DNA Polymerase α

The DNA Polymerase α (Pol α)/primase complex initiates DNA synthesis in eukaryotic replication. In the complex, Pol α and primase cooperate in the production of RNA-DNA oligonucleotides that prime synthesis of new DNA. Here we report crystal structures of the catalytic core of yeast Pol α in unliganded form, bound to an RNA primer/DNA template and extending an RNA primer with deoxynucleotides. We combine the structural analysis with biochemical and computational data to demonstrate that Pol α specifically recognizes the A-form RNA/DNA helix and that the ensuing synthesis of B-form DNA terminates primer synthesis. The spontaneous release of the completed RNA-DNA primer by the Pol α/primase complex simplifies current models of primer transfer to leading- and lagging strand polymerases. The proposed mechanism of nucleotide polymerization by Pol α might contribute to genomic stability by limiting the amount of inaccurate DNA to be corrected at the start of each Okazaki fragment. DOI:http://dx.doi.org/10.7554/eLife.00482.001.

Interplay of DNA polymerase lambda, Protection of Telomeres (Pot) 1 and TERRA in telomere elongation

About 10% – 15% of cancer cell types do not express telomerase, but they use the Alternative Lengthening of Telomeres (ALT) pathway, to counteract telomere shortening. The molecular details of the ALT reaction are poorly characterized and the DNA polymerase (pol) involved in the synthesis step is not known. Here, we show that human DNA pol lambda can actively and efficiently promote annealing telomeric repeats to the complementary C‐rich strand and use it to prime DNA synthesis. DNA pol lambda is associated to telomeres of cells in vivo. Replication protein A negatively regulates this activity, but its inhibition can be counteracted by the Rad9/Hus1/Rad1 complex and the Protection of Telomeres protein 1, which physically interact with and stimulate DNA pol lambda. We show that the telomeric non‐coding RNAs, TERRA can compete with telomeric DNA for annealing and elongation by DNA pol lambda and that Pot1 can counteract this effect. We propose that R‐loops formed by TERRA RNAs at telomeres can function as entry point for the strand invasion during ALT, in a recombination‐independent mechanism. In addition, DNA pol lambda may help to bypass TERRA R‐loops during telomere DNA replication. Supported by an Italian Cancer Research Association AIRC grant.

RNA Polymerase primed DNA synthesis in T7 Bacteriophage system

DNA replication initiation is divided into three steps: Recognition of the replication origin by the initiator protein, melting of the duplex DNA, and synthesis of RNA primer. Primer synthesis is required as there are no known prokaryotic DNA polymerases that can carry out DNA synthesis de novo. In the T7 bacteriophage system and in mitochondrial DNA, the RNA polymerase catalyzes all three steps to initiate DNA replication. The primary replication origin in the T7 genome has two T7 RNA polymerase promoters separated by 52 bp sequence and followed by an AT rich region. There is almost nothing known about the mechanism of RNA‐primed DNA synthesis, including the length of the RNA primer synthesized by T7 RNA polymerase, the mechanism of switching from transcription to DNA synthesis, and why these events occur only at specific promoters in the T7 genome. Whether specific interactions exist between the two polymerases is also not known. Our results suggest that on short DNA substrates, one of the two promoters at the replication origin is sufficient for initiation of DNA synthesis. The RNA polymerase synthesizes primers of 8–12 nucleotide length that are utilized by the DNA polymerase. In the T7 bacteriophage system, this is the critical length at which the transition from transcription initiation to elongation occurs. Our results also show that an actively transcribing RNA polymerase is necessary for the primers to be utilized by the DNA polymerase.Source of research support: NIH

Mutational analysis of the terminal protein Tpg of Streptomyces chromosomes: identification of the deoxynucleotidylation site.

The linear chromosomes and linear plasmids of Streptomyces are capped by terminal proteins (TPs) covalently bound to the 5′ ends of the DNA. The TPs serve as primers for DNA synthesis that patches in the single-stranded gaps at the telomeres resulting from the bi-directional replication (‘end patching’). Typical Streptomyces TPs, designated Tpgs, are conserved in sequence and size (about 185 amino acids), and contain a predicted helix-turn-helix domain and a functional nuclear localization signal. The Tpg-encoding gene (tpg) is often accompanied by an upstream gene tap that encodes an essential telomere-associating protein. Five lone tpg variants (not accompanied by tap) from various Streptomyces species were tested, and three were found to be pseudogenes. The lone tpg variant on the SLP2 plasmid, although functional, still requires the presence of tap on the chromosome for end patching. Using a combination of in vitro deoxynucleotidylation, physical localization, and genetic analysis, we identified the threonine at position 114 (T114) in Tpg of Streptomyces lividans chromosome as the deoxynucleotidylated site. Interestingly, T114 could be substituted by a serine without destroying the priming activity of Tpg in vitro and in vivo. Such T114S substitution is seen in and a number of pseudogenes as well as functional Tpgs. T114 lies in a predicted coil flanked by two short helixes in a highly hydrophilic region. The location and structural arrangement of the deoxynucleotidylated site in Tpg is similar to those in the TPs of phage ø 29 and adenoviruses. However, these TPs are distinct in their sequences and sizes, indicating that they have evolved independently during evolution. Using naturally occurring and artificially created tpg variants, we further identified several amino acid residues in the N-terminus and the helix-turn-helix domain that were important for functionality.