Conserved residues of the C-terminal p16 domain of primase are involved in modulating the activity of the bacterial primosome.
The bacterial primosome comprises the replicative homo-hexameric ring helicase DnaB and the primase DnaG. It is an integral component of the replisome as it unwinds the parental DNA duplex to allow progression of the replication fork, synthesizes the initiation primers at the replication origin, oriC, and the primers required for Okazaki fragment synthesis during lagging strand replication. The interaction between the two component proteins is mediated by a distinct C-terminal domain (p16) of the primase. Both proteins mutually regulate each other's activities and a putative network of conserved residues has been proposed to mediate these effects. We have targeted 10 residues from this network. To investigate the functional contributions of these residues to the primase, ATPase and helicase activities of the primosome, we have used site-directed mutagenesis and in vitro functional assays. Five of these residues (E464, H494, R495, Y548 and R555) exhibited some functional significance while the remaining five (E483, R484, E506, D512 and E530) exhibited no effects. E464 participates in functional modulation of the primase activity, whereas H494, R495 and R555 participate in allosteric functional modulation of the ATPase and/or helicase activities. Y548 contributes directly to the structural interaction with DnaB.
Chintakayala
Kiran
K
Centre for Biomolecular Sciences, School of Chemistry, University of Nottingham, University Park, Nottingham NG7 2RD, UK.
Larson
Marilynn A
MA
Griep
Mark A
MA
Hinrichs
Steven H
SH
Soultanas
Panos
P
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D019915:DNA Primase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism; D053488:DnaB Helicases / Q000235:genetics / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D012329:RNA, Bacterial / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D019915:DNA Primase; D000251:Adenosine Triphosphatases; D053488:DnaB Helicases