Mechanism of DNA Binding by the DnaB Helicase of Escherichia coli:  Analysis of the Roles of Domain γ in DNA Binding

Abstract

We have analyzed the mechanism of single-stranded DNA (ssDNA) binding mediated by the C-terminal domain gamma of the DnaB helicase of Escherichia coli. Sequence analysis of this domain indicated a specific basic region, "RSRARR", and a leucine zipper motif that are likely involved in ssDNA binding. We have carried out deletion as well as in vitro mutagenesis of specific amino acid residues in this region in order to determine their function(s) in DNA binding. The functions of the RSRARR domain in DNA binding were analyzed by site-directed mutagenesis. DnaBMut1, with mutations R(328)A and R(329)A, had a significant decrease in the DNA dependence of ATPase activity and lost its DNA helicase activity completely, indicating the important roles of these residues in DNA binding and helicase activities. DnaBMut2, with mutations R(324)A and R(326)A, had significantly attenuated DNA binding as well as DNA-dependent ATPase and DNA helicase activities, indicating that these residues also play a role in DNA binding and helicase activities. The role(s) of the leucine zipper dimerization motif was (were) determined by deletion analysis. The DnaB Delta 1 mutant with a 55 amino acid C-terminal deletion, which left the leucine zipper and basic DNA binding regions intact, retained DNA binding as well as DNA helicase activities. However, the DnaB Delta 2 mutant with a 113 amino acid C-terminal deletion that included the leucine zipper dimerization motif, but not the RSRARR sequence, lost DNA binding, DNA helicase activities, and hexamer formation. The major findings of this study are (i) the leucine zipper dimerization domain, I(361)-L(389), is absolutely required for (a) dimerization and (b) ssDNA binding; (ii) the base-rich RSRARR sequence is required for DNA binding; (iii) three regions of domain gamma (gamma I, gamma II, and gamma III) differentially regulate the ATPase activity; (iv) there are likely three ssDNA binding sites per hexamer; and (v) a working model of DNA unwinding by the DnaB hexamer is proposed.

The Myxococcus xanthus Developmental Program Can Be Delayed by Inhibition of DNA Replication

“…The ATPase is characterized by two major motifs, a Walker A motif and a Walker B motif, based on comparative analysis of known ATPases (20,37). Domain ␥ (amino acids 310 to 471) contains sites for DNA binding and oligomerization (4). No domain can act alone as a helicase, suggesting that all domains are necessary for this function (3).…”

Mechanisms for initiating cellular DNA replication

“…Adjoining NTDs in the DnaB hexamer self-assemble into a trimer-of-dimers configuration (127)(128)(129)133), creating a collar that binds ssDNA (127). The CTD tier also interacts with nucleic acid substrates but serves as the site for DNA translocation (134)(135)(136)(137). During replication, DnaB encircles the lagging template DNA strand (138), moving 5′→3′ with the ATPase domains oriented toward the duplex side of the fork (138)(139)(140)(141).…”

Roles of Mcm7 and Mcm4 Subunits in the DNA Helicase Activity of the Mouse Mcm4/6/7 Complex

“…DNA binding activities (39). It should be noted that the Nterminal region of Mcm4 contains multiple serine/threonine residues, some of which may be phosphorylation sites of cyclindependent kinase and other kinases.…”