Lipps, Georg
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The monomeric archaeal primase from Nanoarchaeum equitans harbours the features of heterodimeric archaeoeukaryotic primases and primes sequence-specifically
The marine thermophilic archaeon Nanoarchaeum equitans possesses a monomeric primase encompassing the conserved domains of the small catalytic and the large regulatory subunits of archaeoeukaryotic heterodimeric primases in one protein chain. The recombinant protein primes on templates containing a triplet with a central thymidine, thus displaying a pronounced sequence specificity typically observed with bacterial type primases only. The N. equitans primase (NEQ395) is a highly active primase enzyme synthesizing short RNA primers. Termination occurs preferentially at about nine nucleotides, as determined by HPLC analysis and confirmed with mass spectrometry. Possibly, the compact monomeric primase NEQ395 represents the minimal archaeoeukaryotic primase and could serve as a functional and structural model of the heterodimeric archaeoeukaryotic primases, whose study is hindered by engagement in protein assemblies and rather low activity.
Stringent primer termination by an archaeo-eukaryotic DNA primase
Priming of single stranded templates is essential for DNA replication. In recent years, significant progress was made in understanding how DNA primase fulfils this fundamental function, particularly with regard to the initiation. Equally intriguing is the unique property of archeao-eukaryotic primases to terminate primer formation at a well-defined unit length. The apparent ability to “count” the number of bases incorporated prior to primer release is not well understood, different mechanisms having been proposed for different species. We report a mechanistic investigation of primer termination by the pRN1 primase from Sulfolobus islandicus. Using an HPLC-based assay we determined structural features of the primer 5′-end that are required for consistent termination. Mutations within the unstructured linker connecting the catalytic domain to the template binding domain allowed us to assess the effect of altered linker length and flexibility on primer termination.