STRUCTURE AND FUNCTION OF ESCHERICHIA-COLI DNAB PROTEIN - ROLE OF THEN-TERMINAL DOMAIN IN HELICASE ACTIVITY

We have analyzed the contributions of specific domains of DnaB helicas e to its quaternary structure and multienzyme activities. Highly purif ied tryptic fragments containing various domains of DnaB helicase were prepared. Fragment I lacks 14 amino acid (aa) residues from the N-ter minal of DnaB helicase. Fragments II and III are 33-kDa C-terminal and 12-kDa N-terminal polypeptides, respectively, of fragment I. The sing le-stranded DNA-dependent ATPase and DNA helicase activities of DnaB h elicase and its fragments were examined in detail. The ATPase activiti es of native DnaB helicase and fragment I were comparable; however, th e ATPase activity of fragment II was somewhat diminished. Unlike the A TPase activity, the DNA helicase activity was totally abolished in fra gment II and was not complemented by the addition of equimolar fragmen t III. Consequently, the N-terminal 17-kDa domain appeared to have an indispensable role in the DNA helicase action, but not in other enzyma tic activities. Fragment I had a hexameric structure similar to that o bserved with DnaB helicase in both size exclusion HPLC (SE-HPLC) and c hemical cross-linking studies. SE-HPLC analysis indicated that fragmen t II had an apparent hexameric form. However, a detailed chemical cros s-linking analysis showed that it formed stable dimers but the formati on of a stable hexamer was severely impaired. Thus, the N-terminal dom ain appeared to have a strong influence on the hexamer formation. Prot ein sequence analysis indicated that the DnaB protein has a putative ' 'leucine zipper'' [I.X6.L.X6.L.X6.L.X6.L] between amino acid residues 360 and 389 with a consensus basic region located between amino acid r esidues 322 and 331 at the N-terminal, which is a likely site for DNA binding. This site may form an appropriate dimerization site as well a s a DNA binding site in the DnaB helicase.