Helicase-deficient cysteine to glycine substitution mutants of Escherichia coli replication protein PriA retain single-stranded DNA-dependent ATPase activity. Zn2+ stimulation of mutant PriA helicase and primosome assembly activities.

K.H Zavitz,K.J Marians

doi:10.1016/s0021-9258(18)53615-2

K.H Zavitz, K.J Marians

Open Access

https://doi.org/10.1016/s0021-9258(18)53615-2

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Abstract

The PriA replication protein of Escherichia coli guides the ordered assembly of the primosome, the mobile, multiprotein, bidirectional, DNA replication priming/helicase complex of which it is an integral part. Although the PriA protein is not essential for viability, primosome assembly via a PriA-dependent pathway is required for normal cellular replication and growth. The PriA protein itself is multifunctional. In addition to its role in directing primosome assembly, PriA is a site-specific, single-stranded DNA-dependent ATPase (dATPase) and a 3'-->5' DNA translocase and helicase. In an attempt to assess how each individual PriA activity is related to the others (i.e. can one activity function independently of the others or are they intrinsically coupled?), a series of site-directed mutations within priA have been created. priA encodes a cysteine-rich motif, the sequence of which suggests that this region of the protein may be involved in metal binding. Biochemical characterization of three purified cysteine to glycine substitution mutant PriA proteins revealed that these mutant proteins retained their site-specific single-stranded DNA-dependent ATPase activity. However, two of the three mutant proteins were completely incapable of any helicase activity. Residual helicase activity of the third mutant PriA protein could be stimulated 3-fold by the presence of low concentrations of Zn2+ ions. Primosomes assembled with the mutant PriA proteins were also defective in both their ability to act as bidirectional helicase complexes, as well as their ability to synthesize primers for extension by the DNA polymerase III holoenzyme. The results presented here suggest that the cysteine-rich region of PriA is indeed involved in metal binding and that single cysteine to glycine substitutions within this region result in the uncoupling of the ATPase activity of PriA from both its helicase activity and its ability to interact correctly with the DNA template and the six other primosomal proteins.

Highlights

Helicase-deficient Cysteine to Glycine Substitution Mutants of Escherichia coli Replication Protein PriA Retain Single-stranded DNA-dependent ATPaseActivity
The PriA replicationprotein of Escherichiacoli analysis of this replication system revealed that PriA, together guidestheorderedassembly of the primosome, the with six other E. coli proteins, the productsof the priB, priC, mobile, multiprotein,bidirectional, DNA replication d n a T, d m B,dnaC, and dnaG genes, form the primosome [4], priminglhelicasecomplex of which it is an integral a mobile DNA replication priming/helicase complex
With the mutantPriA proteins were defective in direction by r N T P hydrolysis by DnaB [14]

Summary

RESULTS

For the purificationof each protein to apparenthomogeneity, Construction of Mutant Genes and Purification of Mutant Proteins-An examination of the predicted amino acid sequence encoded by priA reveals a cysteine-rich region (8 Cys residues within amino acids)where the spacingof the Cys residues suggests that they may be involved in metal binding (Fig. l), as in the families of so called "zinc-finger," "zincas described previously [24] (data not shown). PriA is stillincreasing and in the linear range even after theamounts of protein were incubated for 10 min at 30 “C in reaction addition of200400 fmol of protein.Similarresults were mixtures containing 18fmol of SSB-coated 6x174 SS(c)DNA as the obtained for the other single C+G mutant, C446G, and the ATPase effector To investigate this apparent difference in the C477G mu- Methods”) was used as a substrate (Fig. 4). Ability to Displace a Short Oligonucleotide from a Heterodu- if Zn2+ions could stimulate the abilitoyf C477G to translocate plex-Since the C-G mutant proteins possessed wild-type extended distances along the single-stranded DawNaAy from levels of PAS-dependent ATPase activity, these proteiwnesre thePAS.Standardreactionmixturessupplemented with tested for their ability to act as DNA helicases. T h e rate of PriA translocation betweenoligomers and 3 3‘- 5‘ directions, in the absence or presence of 10 pM ZnC12

Uncoupling PriA Helicase and ATPase Activities

DISCUSSION

Findings

Uncoupling PriA Helicase and AcTtPivaitsiees