![]() Primer termination is readily observed in primase assays in the absence of further replicative enzymes. Interestingly, the termination of primer synthesis at a defined length is not brought about by a signal from another component of the replisome. Consequently, a mechanism must be in place to terminate the repeated elongation of the primer-template at certain primer lengths. Primases produce a narrow length distribution of primers. This is in contrast to DNA polymerases whose product length is not intrinsically limited and which even use sliding clamps to increase the processivity ( Mizrahi et al., 1985). Typical DNA primases synthesize primers of a defined length, usually between four and 20 nucleotides. Primer synthesis can be divided into two main phases, the first phase being the synthesis of the initial dinucleotide while the second phase is the repeated elongation of the dinucleotide/template ( Kuchta and Stengel, 2010). Archaeal primases that form chimeric or DNA primers ( Beck and Lipps, 2007) thus need to terminate reliably at short primer lengths to prevent errors from affecting genome stability. By using RNA, the replication machinery creates a marker for proofreading of primase-generated stretches during Okazaki fragment processing. Since primases are more error prone than the replicative polymerases ( Sheaff and Kuchta, 1994) the primers have to be replaced after replication by a proofreading enzyme ( Hombauer et al., 2011 Liberti et al., 2013). As a consequence of the unidirectional nature of DNA synthesis, a new primer must be formed for every Okazaki fragment on the lagging strand ( Kornberg, 1980). 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. 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.ĭuring cellular DNA replication, the inability of replicative polymerases to initiate synthesis on a single strand requires the activity of DNA primase. Using an HPLC-based assay we determined structural features of the primer 5′-end that are required for consistent termination. We report a mechanistic investigation of primer termination by the pRN1 primase from Sulfolobus islandicus. 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. Equally intriguing is the unique property of archeao-eukaryotic primases to terminate primer formation at a well-defined unit length. In recent years, significant progress was made in understanding how DNA primase fulfils this fundamental function, particularly with regard to the initiation. Priming of single stranded templates is essential for DNA replication. 3Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland.2Department of Biology, Institute of Biochemistry, ETH Zurich, Zurich, Switzerland.1Institute of Chemistry and Bioanalytics, University of Applied Sciences Northwestern Switzerland, Muttenz, Switzerland.Jan Bergsch 1,2†, Jean-Christophe Devillier 1,2†, Gunnar Jeschke 3 and Georg Lipps 1* ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |