Abstract
Bacterial primase initiates the repeated synthesis of short RNA primers that are extended by DNA polymerase to synthesize Okazaki fragments on the lagging strand at replication forks. It remains unclear how the enzyme recognizes specific initiation sites. In this study, the DnaG primase from Bacillus subtilis (BsuDnaG) was characterized and the crystal structure of the RNA polymerase domain (RPD) was determined. Structural comparisons revealed that the tethered zinc binding domain plays an important role in the interactions between primase and specific template sequence. Structural and biochemical data defined the ssDNA template binding surface as an L shape, and a model for the template ssDNA binding to primase is proposed. The flexibility of the DnaG primases from B. subtilis and G. stearothermophilus were compared, and the results implied that the intrinsic flexibility of the primase may facilitate the interactions between primase and various partners in the replisome. These results shed light on the mechanism by which DnaG recognizes the specific initiation site.
Highlights
In Escherichia coli, the primase (DnaG) transcribes ~2000 to 3000 RNA primers per replication cycle[5]
Biochemical data and structural comparisons revealed that the integrity of zinc binding domain (ZBD) domain in primase is critical for DNA template binding and primer synthesis
No stable RNA polymerase domain (RPD)/ssDNA complex could be detected even at a protein concentration of 3 μM, and the ZBD didn’t bind to ssDNA at all. These results showed that ZBD/RPD still retained DNA binding activity, whereas the deletion of ZBD dramatically affected the DNA binding; the integrity of the ZBD/RPD is significant in ssDNA template binding
Summary
In Escherichia coli, the primase (DnaG) transcribes ~2000 to 3000 RNA primers per replication cycle[5]. To better understand the structure and function of primase and address the interactions between DnaG and specific DNA template, we have carried out structural and functional analysis on DnaG protein from B. subtilis (BsuDnaG). Biochemical data and structural comparisons revealed that the integrity of ZBD domain in primase is critical for DNA template binding and primer synthesis.
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