ABSTRACT
To better characterize the enzymatic activities required for human papillomavirus (HPV) DNA replication, the E1 helicases of HPV types 6 and 11 were produced using a baculovirus expression system. The purified wild type proteins and a version of HPV11 E1 lacking the N-terminal 71 amino acids, which was better expressed, were found to be hexameric over a wide range of concentrations and to have helicase and ATPase activities with relatively low values for K(m)(ATP) of 12 microm for HPV6 E1 and 6 microm for HPV11 E1. Interestingly, the value of K(m)(ATP) was increased 7-fold in the presence of the E2 transactivation domain. In turn, ATP was found to perturb the co-operative binding of E1 and E2 to DNA. Mutant and truncated versions of in vitro translated E1 were used to identify a minimal ATPase domain composed of the C-terminal 297 amino acids. This fragment was expressed, purified, and found to be fully active in ATP hydrolysis, single-stranded DNA binding, and unwinding assays, despite lacking the minimal origin-binding domain.
Subject(s)
Adenosine Triphosphate/metabolism , DNA Helicases/metabolism , Papillomaviridae/enzymology , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , Amino Acid Sequence , Amino Acid Substitution , Base Sequence , Biopolymers , Catalysis , DNA Helicases/chemistry , DNA Primers , Molecular Sequence Data , Papillomaviridae/isolation & purification , Protein Folding , Protein Structure, Quaternary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Substrate SpecificityABSTRACT
A study of 196 renal biopsies was carried out by electron microscopy. In 78 cases concretions were detected, which x-ray microanalysis (EDAX) proved to be calcium phosphate deposits. Concretions always occurred in damaged basement membranes, especially in the basement membranes of Bowman's capsules and of proximal convoluted tubules. They were frequently also found in sclerotic foci. Both nephrotic syndrome (through hypercholesterolaemia) and progressive sclerotic processes seem to play a role in the pathogenesis of concretion formation. Concretions are considered to represent early dystrophic calcification.
