Class-specific restrictions define primase interactions with DNA template and replicative helicase.
Bacterial primase is stimulated by replicative helicase to produce RNA primers that are essential for DNA replication. To identify mechanisms regulating primase activity, we characterized primase initiation specificity and interactions with the replicative helicase for gram-positive Firmicutes (Staphylococcus, Bacillus and Geobacillus) and gram-negative Proteobacteria (Escherichia, Yersinia and Pseudomonas). Contributions of the primase zinc-binding domain, RNA polymerase domain and helicase-binding domain on de novo primer synthesis were determined using mutated, truncated, chimeric and wild-type primases. Key residues in the β4 strand of the primase zinc-binding domain defined class-associated trinucleotide recognition and substitution of these amino acids transferred specificity across classes. A change in template recognition provided functional evidence for interaction in trans between the zinc-binding domain and RNA polymerase domain of two separate primases. Helicase binding to the primase C-terminal helicase-binding domain modulated RNA primer length in a species-specific manner and productive interactions paralleled genetic relatedness. Results demonstrated that primase template specificity is conserved within a bacterial class, whereas the primase-helicase interaction has co-evolved within each species.
Larson
Marilynn A
MA
Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198-5900, USA. malarson@unmc.edu
Griep
Mark A
MA
Bressani
Rafael
R
Chintakayala
Kiran
K
Soultanas
Panos
P
Hinrichs
Steven H
SH
D000595:Amino Acid Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004265:DNA Helicases / Q000378:metabolism*; D019915:DNA Primase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005816:Genetic Complementation Test; D006094:Gram-Positive Bacteria / Q000201:enzymology; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism; D017434:Protein Structure, Tertiary; D020560:Proteobacteria / Q000201:enzymology; D012313:RNA / Q000096:biosynthesis*; D013045:Species Specificity; D013698:Templates, Genetic
D009711:Nucleotides; C034477:RNA primers; D012313:RNA; D004247:DNA; D019915:DNA Primase; D004265:DNA Helicases