Abstract
The T4 gene 59 protein (gp59) serves as an accessory protein to the essential T4-encoded DNA helicase, the gene 41 protein (gp41). gp59 stimulates gp41-dependent DNA synthesis reactions by promoting the assembly of gp41 onto single-stranded DNA (ssDNA), where the enzyme is activated to perform its DNA helicase functions. To better understand the mechanism of helicase-ssDNA assembly, we have studied the effects of gp59 on the intrinsic and ssDNA-stimulated ATPase activities of gp41. Our results indicate that gp59 exerts a direct effect upon the conformation and ATPase activity of gp41, by increasing the affinity of gp41 for ATP. In addition, we find that gp59 is nearly essential for promoting the assembly of gp41 onto ssDNA molecules that are covered with saturating amounts of the T4-encoded helix-destabilizing protein, gene 32 protein (gp32). Results of protein affinity chromatography experiments suggest that gp59 contains distinct binding sites for gp41 and gp32 and may therefore act as a molecular adapter between the helicase and helix-destabilizing proteins. Together, the data indicate that specific gp59-gp41 and gp59-gp32 protein-protein interactions both play important roles in the assembly of the helicase onto single-stranded DNA.
Highlights
The T4 gene 59 protein serves asan accessory upon formation of the primosome, but the gp61 component is protein to the essential T4-encoded DNA helicase, the notessential for helicaseactivity (Venkatesan et al, 1982; gene 41 protein. gp59 stimulates gp41-dependentHinton et al, 1985)
DNA synthesis reactionsby promoting the assembly of gp41 belongs to thefamily of catalytic DNAhelicases (includgp41 onto single-strandedDNA, where theen- ing the dnaBhelicase of Escherichia coli) that unwind duplex zyme is activated to performits DNA helicase functions
Faster thanATP in vitro (Liu and Alberts, 19811, other studies the data indicate that specific gp59-gp41 and gp59-gp3h2ave determined that the enzymheydrolyzes ATP and GTP at protein-protein interactions both play important roles nearly equal rates in vitro (Nossal, 1979).’f3Given the high intheassembly of thehelicaseontosingle-stranded ATP/GTP ratio that exists inside the TCinfected E. coli cell
Summary
1981; Richardson and Nossal, 1989a; Hinton et al, 1987;Alberts et al, 1980;Cha and Albert1s,9881, and resultsof. In experiments involvingvariable protein concentrations, constant salt conditions were maintained between reactions through the addition of appropriate amounts of gp, gp41, and/or gp storage buffers. Samples of purified gp and/or gp, depending on the experiment, were dialyzedinto CB-50 buffer and applied to affinity and control columnsat a flow rate of 2.5 mVh. The columns were washed sequentially with 15-ml steps of buffers CB-50, CB-200, CB-600, and CB-2000 (numerals representing NaCl concentrations in millimoles per liter; all other components identical to CB-50 buffer), while collecting 1.0-mflractions.Aflow rate of 2.5 ml/h was used for the first 7.5 mlof the CB-50 wash step; after that the flow rate was increased to 7.5mVh. Column fractions were analyzed for protein content by Bradford assays and by electrophoresison 15%SDS-polyacrylamide gels. The NaCl concentrations of various column fractions were determined by measuring their relative conductivities on a VWR Scientific model 604 conductivity meter
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
