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 O 2 reduction to H 2 O.

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 0 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.

We report two new findings bearing on the "supranucleo-somal" level of the structure of the Simian Virus 40 minichromosome. I) Isolated SV40 minichromosome which contains all five histones including HI/I/ exists in solution under approximately physiological ionic conditions as a compact roughly spherical particle approximately 300 A in diameter which is capable of fitting within the virus capsid. In spite of such a compact conformation of the minichromosome individual nucleosomes can be readily visualized within the particle. Compact state of SV40 minichromosome depends on both the presence of histone HI and maintenance of approximately physiological ionic strength of solution (micron approximately 0.15). Removal of HI results in a conversion of the compact minichromosomes into an extended (circular beaded) structure. 2) The compact form of the SV40 minichromosome in contract to its circular beaded form is virtually completely resistant to staphylococcal nuclease, strongly suggesting that in particular nuclease-sensitive parts of the internucleosomal DNA regions are not exposed on the outside of the compact SV40 minichromosome. On the other hand, DNase I which is known to attack both inter-and intranucleosomal DNA in the chronatin /2,3/ readily digests the compact form of the SV40 minichromosome. Possible models of the compact minichromosome and implications for higher order structures of the cellular chromatin are discussed.

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.

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