Structure and function of Escherichia coli DnaB protein: role of the N-terminal domain in helicase activity.
We have analyzed the contributions of specific domains of DnaB helicase to its quaternary structure and multienzyme activities. Highly purified tryptic fragments containing various domains of DnaB helicase were prepared. Fragment I lacks 14 amino acid (aa) residues from the N-terminal of DnaB helicase. Fragments II and III are 33-kDa C-terminal and 12-kDa N-terminal polypeptides, respectively, of fragment I. The single-stranded DNA-dependent ATPase and DNA helicase activities of DnaB helicase and its fragments were examined in detail. The ATPase activities of native DnaB helicase and fragment I were comparable; however, the ATPase activity of fragment II was somewhat diminished. Unlike the ATPase activity, the DNA helicase activity was totally abolished in fragment II and was not complemented by the addition of equimolar fragment III. Consequently, the N-terminal 17-kDa domain appeared to have an indispensable role in the DNA helicase action, but not in other enzymatic activities. Fragment I had a hexameric structure similar to that observed with DnaB helicase in both size exclusion HPLC (SE-HPLC) and chemical cross-linking studies. SE-HPLC analysis indicated that fragment II had an apparent hexameric form. However, a detailed chemical cross-linking analysis showed that it formed stable dimers but the formation of a stable hexamer was severely impaired. Thus, the N-terminal domain appeared to have a strong influence on the hexamer formation.(ABSTRACT TRUNCATED AT 250 WORDS)
Biswas
S B
SB
Department of Pediatrics, University of Maryland School of Medicine, Baltimore 21201, USA.
Chen
P H
PH
Biswas
E E
EE
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004265:DNA Helicases / Q000737:chemistry* / Q000378:metabolism*; D053488:DnaB Helicases; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D016350:Leucine Zippers; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011487:Protein Conformation; D014357:Trypsin
D001426:Bacterial Proteins; D010446:Peptide Fragments; D014357:Trypsin; D000251:Adenosine Triphosphatases; C506730:dnaB protein, E coli; D004265:DNA Helicases; D053488:DnaB Helicases