Abstract
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.
Highlights
During cellular DNA replication, the inability of replicative polymerases to initiate synthesis on a single strand requires the activity of DNA primase
Its function is the de novo synthesis of oligonucleotides on single stranded DNA serving as a substrate for a DNA polymerase that in turn extends the primer at its 3 -OH terminus
Point mutants were generated according to the QuickChange PCR protocol involving two mutagenic oligonucleotide primers and Pfu polymerase, parental DNA was digested with DpnI
Summary
During cellular DNA replication, the inability of replicative polymerases to initiate synthesis on a single strand requires the activity of DNA primase. This mode of termination has been proposed for the human primosome based on structural evidence of sterical hindrance between p58N and p58C leading to a clash exactly after 9 nt (Baranovskiy et al, 2016a). We investigated which of the proposed mechanisms of primer termination could apply to pRN1 primase To this end, we took advantage of the covalent coupling of the catalytic domain and the HBD which allowed us to rigorously test the mechanistic requirements of primer termination by constructing mutants with linker sequences differing in length and flexibility. We built a structure model of pRN1 primase in complex with the substrate DNA
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