Nucleic Acid Chaperone Activity Research Articles

DNA Interaction Kinetics of HIV-1 Nucleocapsid and Gag Proteins

The human immunodeficiency virus type 1 (HIV-1) Gag protein is essential for retroviral assembly. During viral maturation, Gag is processed to form matrix, capsid, and nucleocapsid (NC). NC is initially cleaved into the larger NCp15, then to NCp9, and finally to NCp7. NCp7 functions as a nucleic acid chaperone during retroviral replication, in which it rearranges nucleic acids to facilitate reverse transcription and recombination. The role of Gag and intermediate forms of NC in facilitating nucleic acid remodeling is not well understood, although it is likely that they also function as chaperones during viral assembly and early steps of reverse transcription. To investigate the capability of Gag and precursor forms of NC to act as nucleic acid chaperones, we use single DNA molecule stretching to probe how these proteins alter DNA aggregation, duplex destabilization, and DNA interaction kinetics. These characteristics are critical for efficient nucleic acid chaperone activity. Duplex annealing in the presence of NCp7 indicates that this protein dissociates rapidly from single-stranded DNA. In contrast, Gag inhibits DNA annealing, as indicated by strong hysteresis observed when stretching and relaxing DNA in the presence of Gag. We use a new method to measure the rate at which DNA that has been melted by force is able to reanneal in the presence of Gag and NC proteins. The results show that DNA annealing in the presence of Gag occurs on the time scale of minutes, compared to ∼1 second for annealing in the presence of NCp7. Further studies of reannealing kinetics in the presence of NCp9, NCp15, and Gag deletion constructs will elucidate the role of specific protein domains in determining Gag- and NC-DNA interaction kinetics.

Nucleic-acid-binding properties of the C2-L1Tc nucleic acid chaperone encoded by L1Tc retrotransposon

It has been reported previously that the C2-L1Tc protein located in the Trypanosoma cruzi LINE (long interspersed nuclear element) L1Tc 3′ terminal end has NAC (nucleic acid chaperone) activity, an essential activity for retrotransposition of LINE-1. The C2-L1Tc protein contains two cysteine motifs of a C2H2 type, similar to those present in TFIIIA (transcription factor IIIA). The cysteine motifs are flanked by positively charged amino acid regions. The results of the present study show that the C2-L1Tc recombinant protein has at least a 16-fold higher affinity for single-stranded than for double-stranded nucleic acids, and that it exhibits a clear preference for RNA binding over DNA. The C2-L1Tc binding profile (to RNA and DNA) corresponds to a non-co-operative-binding model. The zinc fingers present in C2-L1Tc have a different binding affinity to nucleic acid molecules and also different NAC activity. The RRR and RRRKEK [NLS (nuclear localization sequence)] sequences, as well as the C2H2 zinc finger located immediately downstream of these basic stretches are the main motifs responsible for the strong affinity of C2-L1Tc to RNA. These domains also contribute to bind single- and double-stranded DNA and have a duplex-stabilizing effect. However, the peptide containing the zinc finger situated towards the C-terminal end of C2-L1Tc protein has a slight destabilization effect on a mismatched DNA duplex and shows a strong preference for single-stranded nucleic acids, such as C2-L1Tc. These results provide further insight into the essential properties of the C2-L1Tc protein as a NAC.

DNA Interaction Properties of Nucleic Acid Chaperone Proteins from Retrotransposons

Nucleic acid chaperone activity is an essential component of reverse transcription in retroviruses and retrotransposons. Using DNA stretching with optical tweezers, we have developed a method for detailed characterization of nucleic acid chaperone proteins, which facilitate the rearrangement of nucleic acid secondary structure. The nucleic acid chaperone properties of the human immunodeficiency virus type-1 (HIV-1) nucleocapsid protein (NC) have been extensively studied, and duplex destabilization, nucleic acid aggregation, and rapid protein binding kinetics have been identified as major components of its activity. The chaperone properties of other nucleic acid chaperone proteins, such as those from the retrotransposons LINE-1 and Ty3, ORF1p and Ty3 NC, are not well understood. We used single molecule DNA stretching to characterize the activity of wild type and mutant ORF1p and Ty3 NC. ORF1p binds both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) with high affinity, and strongly aggregates both forms. It is therefore an excellent chaperone, and altering certain residues has dramatic effects on chaperone activity. Wild type Ty3 also strongly aggregates both dsDNA and ssDNA, and melted DNA exhibits more rapid reannealing in the presence of Ty3 NC, relative to that observed in the presence of ORF1p. We examine several Ty3 NC mutants to identify the roles of functional regions of the protein in its chaperone activity. This research was supported in part by funding from INSERM and ANRS (France).

Regulation of the nucleic acid chaperone activity of HTLV-1 Nucleocapsid Protein

Nucleocapsid proteins (NC) of retroviruses are nucleic acid chaperones that facilitate nucleic acid remodeling. This property of NC proteins is critical for their role in viral genome dimerization, maturation and reverse transcription. In contrast to all other NC proteins studied to date, the human T-cell leukemia virus type 1 (HTLV-1) NC protein was shown to be an extremely poor chaperone. In this work, we demonstrate that the anionic C-terminal domain (CTD) of this protein is responsible for its poor chaperone function. Single molecule DNA stretching studies suggest that HTLV-1 NC dissociates very slowly from single-stranded DNA, which may be a primary reason for its poor chaperone activity. In contrast, a truncation mutant that lacks the CTD is a more effective annealing agent and displays faster off-rate kinetics. Under conditions of high ionic strength, the properties of the WT and CTD-deletion variant are much more similar to each other. Taken together, our data suggest that an electrostatic attraction between the anionic CTD and cationic N-terminal domain of HTLV-1 NC leads to polymerization onto ssDNA resulting in a poor ability to aggregate nucleic acids or to promote their annealing. This property of HTLV-1 NC makes it similar to typical SSB proteins, and may be related to this NC's role in excluding the viral restriction factor APOBEC3G from HTLV particles.

Fidelity of plus-strand priming requires the nucleic acid chaperone activity of HIV-1 nucleocapsid protein

During minus-strand DNA synthesis, RNase H degrades viral RNA sequences, generating potential plus-strand DNA primers. However, selection of the 3′ polypurine tract (PPT) as the exclusive primer is required for formation of viral DNA with the correct 5′-end and for subsequent integration. Here we show a new function for the nucleic acid chaperone activity of HIV-1 nucleocapsid protein (NC) in reverse transcription: blocking mispriming by non-PPT RNAs. Three representative 20-nt RNAs from the PPT region were tested for primer extension. Each primer had activity in the absence of NC, but less than the PPT. NC reduced priming by these RNAs to essentially base-line level, whereas PPT priming was unaffected. RNase H cleavage and zinc coordination by NC were required for maximal inhibition of mispriming. Biophysical properties, including thermal stability, helical structure and reverse transcriptase (RT) binding affinity, showed significant differences between PPT and non-PPT duplexes and the trends were generally correlated with the biochemical data. Binding studies in reactions with both NC and RT ruled out a competition binding model to explain NC's observed effects on mispriming efficiency. Taken together, these results demonstrate that NC chaperone activity has a major role in ensuring the fidelity of plus-strand priming.

Effect of Mg 2+ and Na + on the Nucleic Acid Chaperone Activity of HIV-1 Nucleocapsid Protein: Implications for Reverse Transcription

The human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein (NC) is an essential protein for retroviral replication. Among its numerous functions, NC is a nucleic acid (NA) chaperone protein that catalyzes NA rearrangements leading to the formation of thermodynamically more stable conformations. In vitro, NC chaperone activity is typically assayed under conditions of low or no Mg 2+, even though reverse transcription requires the presence of divalent cations. Here, the chaperone activity of HIV-1 NC was studied as a function of varying Na + and Mg 2+ concentrations by investigating the annealing of complementary DNA and RNA hairpins derived from the trans-activation response domain of the HIV genome. This reaction mimics the annealing step of the minus-strand transfer process in reverse transcription. Gel-shift annealing and sedimentation assays were used to monitor the annealing kinetics and aggregation activity of NC, respectively. In the absence of protein, a limited ability of Na + and Mg 2+ cations to facilitate hairpin annealing was observed, whereas NC stimulated the annealing 10 3- to 10 5-fold. The major effect of either NC or the cations is on the rate of bimolecular association of the hairpins. This effect is especially strong under conditions wherein NC induces NA aggregation. Titration with NC and NC/Mg 2+ competition studies showed that the annealing kinetics depends only on the level of NA saturation with NC. NC competes with Mg 2+ or Na + for sequence-nonspecific NA binding similar to a simple trivalent cation. Upon saturation, NC induces attraction between NA molecules corresponding to ∼ 0.3 kcal/mol/nucleotide, in agreement with an electrostatic mechanism of NC-induced NA aggregation. These data provide insights into the variable effects of NC's chaperone activity observed during in vitro studies of divalent metal-dependent reverse transcription reactions and suggest the feasibility of NC-facilitated proviral DNA synthesis within the mature capsid core.

Applications of Nucleic Acid Chaperone Activity of CspA and Its Homologues

In Escherichia coli, the cold shock response is exerted upon temperature change from 37 to 15°C and is characterized by induction of several cold shock proteins including its major cold shock protein, CspA. E. coli CspA family consists of nine members, CspA to CspI. CspA and some of its homologues play a critical role in cold acclimation of cells as RNA chaperones by destabilizing secondary structures in RNAs. Here, we showed that the nucleic acid melting activity of Csp proteins can be used to facilitate reactions, such as RT-PCR or RNA cleavage reactions by endoribonucleases, which are hindered by presence of secondary structures in the DNA/RNA substrate used. The low substrate specificity of Csps together with their compatibility with various enzymes and their stability and activity over a broad temperature range makes them ideal candidates to be used for a variety of processes.

A single amino acid substitution in ORF1 dramatically decreases L1 retrotransposition and provides insight into nucleic acid chaperone activity

L1 is a ubiquitous interspersed repeated sequence in mammals that achieved its high copy number by autonomous retrotransposition. Individual L1 elements within a genome differ in sequence and retrotransposition activity. Retrotransposition requires two L1-encoded proteins, ORF1p and ORF2p. Chimeric elements were used to map a 15-fold difference in retrotransposition efficiency between two L1 variants from the mouse genome, TFC and TFspa, to a single amino acid substitution in ORF1p, D159H. The steady-state levels of L1 RNA and protein do not differ significantly between these two elements, yet new insertions are detected earlier and at higher frequency in TFC, indicating that it converts expressed L1 intermediates more effectively into new insertions. The two ORF1 proteins were purified and their nucleic acid binding and chaperone activities were examined in vitro. Although the RNA and DNA oligonucleotide binding affinities of these two ORF1 proteins were largely indistinguishable, D159 was significantly more effective as a nucleic acid chaperone than H159. These findings support a requirement for ORF1p nucleic acid chaperone activity at a late step during L1 retrotransposition, extend the region of ORF1p that is known to be critical for its functional interactions with nucleic acids, and enhance understanding of nucleic acid chaperone activity.

Dissecting CNBP, a Zinc-Finger Protein Required for Neural Crest Development, in Its Structural and Functional Domains

Cellular nucleic-acid-binding protein (CNBP) plays an essential role in forebrain and craniofacial development by controlling cell proliferation and survival to mediate neural crest expansion. CNBP binds to single-stranded nucleic acids and displays nucleic acid chaperone activity in vitro. The CNBP family shows a conserved modular organization of seven Zn knuckles and an arginine-glycine-glycine (RGG) box between the first and second Zn knuckles. The participation of these structural motifs in CNBP biochemical activities has still not been addressed. Here, we describe the generation of CNBP mutants that dissect the protein into regions with structurally and functionally distinct properties. Mutagenesis approaches were followed to generate: (i) an amino acid replacement that disrupted the fifth Zn knuckle; (ii) N-terminal deletions that removed the first Zn knuckle and the RGG box, or the RGG box alone; and (iii) a C-terminal deletion that eliminated the three last Zn knuckles. Mutant proteins were overexpressed in Escherichia coli, purified, and used to analyze their biochemical features in vitro, or overexpressed in Xenopus laevis embryos to study their function in vivo during neural crest cell development. We found that the Zn knuckles are required, but not individually essential, for CNBP biochemical activities, whereas the RGG box is essential for RNA–protein binding and nucleic acid chaperone activity. Removal of the RGG box allowed CNBP to preserve a weak single-stranded-DNA-binding capability. A mutant mimicking the natural N-terminal proteolytic CNBP form behaved as the RGG-deleted mutant. By gain-of-function and loss-of-function experiments in Xenopus embryos, we confirmed the participation of CNBP in neural crest development, and we demonstrated that the CNBP mutants lacking the N-terminal region or the RGG box alone may act as dominant negatives in vivo. Based on these data, we speculate about the existence of a specific proteolytic mechanism for the regulation of CNBP biochemical activities during neural crest development.

Deaminase-independent inhibition of HIV-1 reverse transcription by APOBEC3G

APOBEC3G (A3G), a host protein that inhibits HIV-1 reverse transcription and replication in the absence of Vif, displays cytidine deaminase and single-stranded (ss) nucleic acid binding activities. HIV-1 nucleocapsid protein (NC) also binds nucleic acids and has a unique property, nucleic acid chaperone activity, which is crucial for efficient reverse transcription. Here we report the interplay between A3G, NC and reverse transcriptase (RT) and the effect of highly purified A3G on individual reactions that occur during reverse transcription. We find that A3G did not affect the kinetics of NC-mediated annealing reactions, nor did it inhibit RNase H cleavage. In sharp contrast, A3G significantly inhibited all RT-catalyzed DNA elongation reactions with or without NC. In the case of (−) strong-stop DNA synthesis, the inhibition was independent of A3G's catalytic activity. Fluorescence anisotropy and single molecule DNA stretching analyses indicated that NC has a higher nucleic acid binding affinity than A3G, but more importantly, displays faster association/disassociation kinetics. RT binds to ssDNA with a much lower affinity than either NC or A3G. These data support a novel mechanism for deaminase-independent inhibition of reverse transcription that is determined by critical differences in the nucleic acid binding properties of A3G, NC and RT.

Single-Molecule Study of the Inhibition of HIV-1 Transactivation Response Region DNA/DNA Annealing by Argininamide

Single-molecule spectroscopy was used to examine how a model inhibitor of HIV-1, argininamide, modulates the nucleic acid chaperone activity of the nucleocapsid protein (NC) in the minus-strand transfer step of HIV-1 reverse transcription, in vitro. In minus-strand transfer, the transactivation response region (TAR) RNA of the genome is annealed to the complementary "TAR DNA" generated during minus-strand strong-stop DNA synthesis. Argininamide and its analogs are known to bind to the hairpin bulge region of TAR RNA as well as to various DNA loop structures, but its ability to inhibit the strand transfer process has only been implied. Here, we explore how argininamide modulates the annealing kinetics and secondary structure of TAR DNA. The studies reveal that the argininamide inhibitory mechanism involves a shift of the secondary structure of TAR, away from the NC-induced "Y" form, an intermediate in reverse transcription, and toward the free closed or "C" form. In addition, more potent inhibition of the loop-mediated annealing pathway than stem-mediated annealing is observed. Taken together, these data suggest a molecular mechanism wherein argininamide inhibits NC-facilitated TAR RNA/DNA annealing in vitro by interfering with the formation of key annealing intermediates.

Effects of nucleic acid local structure and magnesium ions on minus-strand transfer mediated by the nucleic acid chaperone activity of HIV-1 nucleocapsid protein

HIV-1 nucleocapsid protein (NC) is a nucleic acid chaperone, which is required for highly specific and efficient reverse transcription. Here, we demonstrate that local structure of acceptor RNA at a potential nucleation site, rather than overall thermodynamic stability, is a critical determinant for the minus-strand transfer step (annealing of acceptor RNA to (−) strong-stop DNA followed by reverse transcriptase (RT)-catalyzed DNA extension). In our system, destabilization of a stem-loop structure at the 5′ end of the transactivation response element (TAR) in a 70-nt RNA acceptor (RNA 70) appears to be the major nucleation pathway. Using a mutational approach, we show that when the acceptor has a weak local structure, NC has little or no effect. In this case, the efficiencies of both annealing and strand transfer reactions are similar. However, when NC is required to destabilize local structure in acceptor RNA, the efficiency of annealing is significantly higher than that of strand transfer. Consistent with this result, we find that Mg2+ (required for RT activity) inhibits NC-catalyzed annealing. This suggests that Mg2+ competes with NC for binding to the nucleic acid substrates. Collectively, our findings provide new insights into the mechanism of NC-dependent and -independent minus-strand transfer.

Single DNA molecule stretching measures the activity of chemicals that target the HIV-1 nucleocapsid protein

We develop a biophysical method for investigating chemical compounds that target the nucleic acid chaperone activity of HIV-1 nucleocapsid protein (NCp7). We used an optical tweezers instrument to stretch single λ-DNA molecules through the helix–coil transition in the presence of NCp7 and various chemical compounds. The change in the helix–coil transition width induced by wild-type NCp7 and its zinc finger variants correlates with in vitro nucleic acid chaperone activity measurements and in vivo assays. The compound–NC interaction measured here reduces NCp7’s capability to alter the transition width. Purified compounds from the NCI Diversity set, 119889, 119911, and 119913 reduce the chaperone activity of 5 nM NC in aqueous solution at 10, 25, and 100 nM concentrations respectively. Similarly, gallein reduced the activity of 4 nM NC at 100 nM concentration. Further analysis allows us to dissect the impact of each compound on both sequence-specific and non-sequence-specific DNA binding of NC, two of the main components of NC’s nucleic acid chaperone activity. These results suggest that DNA stretching experiments can be used to screen chemical compounds targeting NC proteins and to further explore the mechanisms by which these compounds interact with NC and alter its nucleic acid chaperone activity.

Rapid Kinetics of Protein–Nucleic Acid Interaction is a Major Component of HIV-1 Nucleocapsid Protein’s Nucleic Acid Chaperone Function

The nucleic acid chaperone activity of the human immunodeficiency virus type-1 (HIV-1) nucleocapsid protein (NC) plays an important role in the retroviral life cycle, in part, by facilitating numerous nucleic acid rearrangements throughout the reverse transcription process. Recent studies have identified duplex destabilization and nucleic acid aggregation as the two major components of NC's chaperone activity. In order to better understand the contribution of the functional domains of NC to these two activities, we used optical tweezers to stretch single lambda DNA molecules through the helix-coil transition in the presence of wild-type or mutant HIV-1 NC. Protein-induced duplex destabilization was measured directly as an average decrease of the force-induced melting free energy, while NC's ability to facilitate strand annealing was determined by the amount of hysteresis in the DNA stretch-relax cycle. By studying zinc-free variants of full-length and truncated NC, the relative contributions of NC's zinc fingers and N-terminal basic domain to the two major components of chaperone activity were elucidated. In addition, examination of NC variants containing mutations affecting one or both zinc finger motifs showed that effective strand annealing activity is correlated with NC's ability to rapidly bind and dissociate from nucleic acids. NC variants with slow on/off rates are inefficient in strand annealing, even though they may still be capable of high affinity nucleic acid binding, duplex destabilization, and/or nucleic acid aggregation. Taken together, these observations establish the rapid kinetics of protein–nucleic acid interaction as another major component of NC's chaperone function.

Mechanistic Studies of Mini-TAR RNA/DNA Annealing in the Absence and Presence of HIV-1 Nucleocapsid Protein

HIV-1 reverse transcription involves several nucleic acid rearrangements, which are catalyzed by the nucleocapsid protein (NC). Annealing of the trans-activation response element (TAR) DNA hairpin to a complementary TAR RNA hairpin, resulting in the formation of an extended 98 –base-pair duplex, is an essential step in the minus-strand transfer step of reverse transcription. To elucidate the TAR RNA/DNA annealing reaction pathway, annealing kinetics were studied systematically by gel-shift assays performed in the presence or absence of HIV-1 NC. Truncated 27 nucleotide mini-TAR RNA and DNA constructs were used in this work. In the absence of NC, the annealing is slow, and involves the fast formation of an unstable extended “kissing” loop intermediate, followed by a slower strand exchange between the terminal stems. This annealing is very sensitive to loop–loop complementarity, as well as to nucleic acid concentration, ionic strength and temperature. NC stimulates the annealing ∼ 5000-fold by stabilizing the bimolecular intermediate ∼ 100 to 200-fold, and promoting the subsequent strand exchange reaction ∼ 10 to 20-fold. NC concentration dependence studies suggest that there is a direct correlation between the amount of NC required to stabilize the intermediate and the amount needed to induce mini-TAR aggregation. Whereas saturating levels of NC are required to efficiently aggregate nucleic acids, sub-saturating NC is sufficient to significantly enhance duplex destabilization. Equilibrium levels of mini-TAR RNA/DNA annealing were also measured under a variety of conditions. Taken together, the results presented here provide a quantitative accounting of HIV-1 NC's aggregation and duplex destabilizing activity, and provide insights into the universal nucleic acid chaperone activity of this essential viral protein.

APOBEC3 Proteins Inhibit Human LINE-1 Retrotransposition

The human cytidine deaminase family APOBEC3 represents a novel group of proteins in the field of innate defense mechanisms that has been shown to be active against a variety of retroviruses. Here we examined whether members of the APO-BEC3 family have an impact on retrotransposition of human long interspersed nuclear elements (LINE-1s or L1s). Using a retrotransposition reporter assay in HeLa cells, we demonstrate that in the presence of transiently transfected APOBEC3A, L1 retrotransposition frequency was reduced by up to 85%. Although APOBEC3G and -3H did not influence L1 retrotransposition notably, expression of APOBEC3B, -3C, and -3F inhibited transposition by approximately 75%. Although reverse transcription of L1s occurs in the nucleus and APOBEC3 proteins are believed to act via DNA deamination during reverse transcription, activity against L1 retrotransposition was not correlated with nuclear localization of APOBEC3s. We demonstrate that APOBEC3C and APOBEC3B were endogenously expressed in HeLa cells. Accordingly, down-regulation of APOBEC3C by RNA interference enhanced L1 retrotransposition by approximately 78%. Sequence analyses of de novo L1 retrotransposition events that occurred in the presence of overexpressed APOBEC3 proteins as well as the analyses of pre-existing endogenous L1 elements did not reveal an enhanced rate of G-to-A transitions, pointing to a mechanism independent of DNA deamination. This study presents evidence for a role of host-encoded APOBEC3 proteins in the regulation of L1 retrotransposition.

Poliovirus Protein 3AB Displays Nucleic Acid Chaperone and Helix-Destabilizing Activities

Poliovirus protein 3AB displayed nucleic acid chaperone activity in promoting the hybridization of complementary nucleic acids and destabilizing secondary structure. Hybridization reactions at 30 degrees C between 20- and 40-nucleotide RNA oligonucleotides and 179- or 765-nucleotide RNAs that contained a complementary region were greatly enhanced in the presence of 3AB. The effect was nonspecific as reactions between DNA oligonucleotides and RNA or DNA templates were also enhanced. Reactions were optimal with 1 mM MgCl(2) and 20 mM KCl. Analysis of the reactions with various 3AB and template concentrations indicated that enhancement required a critical amount of 3AB that increased as the concentration of nucleic acid increased. This was consistent with a requirement for 3AB to "coat" the nucleic acids for enhancement. The helix-destabilizing activity of 3AB was tested in an assay with two 42-nucleotide completely complementary DNAs. Each complement formed a strong stem-loop (DeltaG = -7.2 kcal/mol) that required unwinding for hybridization to occur. DNAs were modified at the 3' or 5' end with fluorescent probes such that hybridization resulted in quenching of the fluorescent signal. Under optimal conditions at 30 degrees C, 3AB stimulated hybridization in a concentration-dependent manner, as did human immunodeficiency virus nucleocapsid protein, an established chaperone. The results are discussed with respect to the role of 3AB in viral replication and recombination.

Spatial Assembly and RNA Binding Stoichiometry of a LINE-1 Protein Essential for Retrotransposition

LINE-1, or L1, is a highly successful retrotransposon in mammals, comprising 17% and 19% of the human and mouse genomes, respectively. L1 retrotransposition and hence amplification requires the protein products of its two open reading frames, ORF1 and ORF2. The sequence of the ORF1 protein (ORF1p) is not related to any protein with known function. ORF1p has RNA binding and nucleic acid chaperone activities that are both required for retrotransposition. Earlier studies have shown that ORF1p forms a homotrimer with an asymmetric dumbbell shape, in which a rod separates a large end from a small end. Here, we determine the topological arrangement of monomers within the homotrimer by comparing atomic force microscopy (AFM) images of the full ORF1p with those of truncations containing just the N or C-terminal regions. In addition, AFM images of ORF1p bound to RNA at high protein/RNA molar ratios show that ORF1p can form tightly packed clusters on RNA, with binding occurring at the C-terminal domain. The number of bound ORF1p trimers increases with increasing length of the RNA, revealing that the binding site size is about 50 nt, a value confirmed by nitrocellulose filter binding under stoichiometric conditions. These results are consistent with a role for ORF1p during L1 retrotransposition that includes both coating the RNA and acting as a nucleic acid chaperone. Furthermore, these in vitro L1 ribonucleoprotein particles provide insight into the structure of the L1 retrotransposition intermediate.

LINE-1 Retrotransposition: Impact on Genome Stability and Diversity and Human Disease

LINE-1 Retrotransposition: Impact on Genome Stability and Diversity and Human Disease

The ORF1 Protein Encoded by LINE‐1: Structure and Function During L1 Retrotransposition

LINE-1, or L1 is an autonomous non-LTR retrotransposon in mammals. Retrotransposition requires the function of the two, L1-encoded polypeptides, ORF1p and ORF2p. Early recognition of regions of homology between the predicted amino acid sequence of ORF2 and known endonuclease and reverse transcriptase enzymes led to testable hypotheses regarding the function of ORF2p in retrotransposition. As predicted, ORF2p has been demonstrated to have both endonuclease and reverse transcriptase activities. In contrast, no homologs of known function have contributed to our understanding of the function of ORF1p during retrotransposition. Nevertheless, significant advances have been made such that we now know that ORF1p is a high affinity RNA binding protein that forms a ribonucleoprotein particle together with L1 RNA. Furthermore, ORF1p is a nucleic acid chaperone and this nucleic acid chaperone activity is required for L1 retrotransposition.