Search

The human LINE-1/L1 ORF2 protein is a multifunctional enzyme which plays a vital role in the life cycle of the human L1 retrotransposon. The protein consists of an endonuclease domain, followed by a central reverse transcriptase domain and a carboxy-terminal C-domain with unknown function. Here, we explore the nucleic acid binding properties of the 180-amino acid carboxy-terminal segment (CTS) of the human L1 ORF2p in vitro. In a series of experiments involving gel shift assay, we demonstrate that the CTS of L1 ORF2p binds RNA in non-sequence-specific manner. Finally, we report that mutations destroying the putative Zn-knuckle structure of the protein do not significantly affect the level of RNA binding and discuss the possible functional role of the CTS in L1 retrotransposition., Highlights • The 180-aa C-terminal segment (CTS) of human L1 ORF2p was expressed and purified from bacteria. • The nucleic acid binding properties of the CTS of L1ORF2p were examined in vitro. • The CTS of L1 ORF2p is an RNA binding domain. • The CTS of L1 ORF2p binds RNA in non-sequence-specific manner in the low nanomolar range. • Disruption of the putative Zn-knuckle structure does not significantly affect RNA binding.

L1 elements (LINE-1s) account for 17% of the human genome and have achieved this abundance by transpositions via an RNA intermediate, or retrotransposition. Reverse transcription is a crucial event in the retrotransposition of the active human L1 element and is carried out by the L1-encoded ORF2 protein. Previously, we performed biochemical characterization of the human L1 ORF2 protein with reverse transcriptase (RT) activity (referred to as L1 RT), expressed in baculovirus-infected insect cells. In the present study, we describe the properties of DNA- and RNA-dependent DNA synthesis catalyzed by the L1 RT on the L1 templates in vitro. We found that L1 RT synthesized at least 620 of nucleotides per template binding event utilizing L1 RNA in vitro. Under processive conditions the L1 RT synthesized cDNA over 5 times longer than that Moloney murine leukemia virus RT on the heteropolymeric RNA template used in these studies. These data are the first to demonstrate that RT from the human L1 element is a highly processive polymerase among RT enzymes. This report also presents a strong evidence of lack of RNase H activity for the L1 ORF2 protein in vitro, distinguishing L1 RT from retroviral RTs. Finally, we found strong pausing for of the L1 RT during DNA polymerization within the 3′ untranslated region of L1 mRNA, that is result of contribution both rGs runs of the polypurine stretch and immediately adjacent stem–loop structure. A mechanism facilitating minus-strand DNA synthesis during reverse transcription of L1 element in vivo is discussed.

Background Laccases belong to multicopper oxidases, a widespread class of enzymes implicated in many oxidative functions in pathogenesis, immunogenesis and morphogenesis of organisms and in the metabolic turnover of complex organic substances. They catalyze the coupling between the four one-electron oxidations of a broad range of substrates with the four-electron reduction of dioxygen to water. These catalytic processes are made possible by the contemporaneous presence of at least four copper ion sites, classified according to their spectroscopic properties: one type 1 (T1) site where the electrons from the reducing substrates are accepted, one type 2 (T2), and a coupled binuclear type 3 pair (T3) which are assembled in a T2/T3 trinuclear cluster where the electrons are transferred to perform the O2 reduction to H2O. Results The structure of a laccase from the white-rot fungus Lentinus (Panus) tigrinus, a glycoenzyme involved in lignin biodegradation, was solved at 1.5 Å. It reveals a asymmetric unit containing two laccase molecules (A and B). The progressive reduction of the copper ions centers obtained by the long-term exposure of the crystals to the high-intensity X-ray synchrotron beam radiation under aerobic conditions and high pH allowed us to detect two sequential intermediates in the molecular oxygen reduction pathway: the "peroxide" and the "native" intermediates, previously hypothesized through spectroscopic, kinetic and molecular mechanics studies. Specifically the electron-density maps revealed the presence of an end-on bridging, μ-η1:η1 peroxide ion between the two T3 coppers in molecule B, result of a two-electrons reduction, whereas in molecule A an oxo ion bridging the three coppers of the T2/T3 cluster (μ3-oxo bridge) together with an hydroxide ion externally bridging the two T3 copper ions, products of the four-electrons reduction of molecular oxygen, were best modelled. Conclusion This is the first structure of a multicopper oxidase which allowed the detection of two intermediates in the molecular oxygen reduction and splitting. The observed features allow to positively substantiate an accurate mechanism of dioxygen reduction catalyzed by multicopper oxidases providing general insights into the reductive cleavage of the O-O bonds, a leading problem in many areas of biology.