Chemically modified DNA substrates implicate the importance of electrostatic interactions for DNA unwinding by Dda helicase.
Helicase-catalyzed disruption of double-stranded nucleic acid is vital to DNA replication, recombination, and repair in all forms of life. The relative influence of specific chemical interactions between helicase and the substrate over a series of multistep catalytic events is still being defined. To this end, three modified DNA oligonucleotides were designed to serve as substrates for the bacteriophage T4 helicase, Dda. A 5'-DNA-PNA-DNA-3' chimera was synthesized, thereby, conferring both a loss of charge and altering the conformational flexibility of the oligonucleotide. The second modified oligonucleotide possessed a single methylphosphonate replacement on the phosphate backbone, creating a gap in the charge distribution of the substrate. The third modification introduced an abasic site into the oligonucleotide sequence. This abasic site retains the charge distribution of the normal DNA substrate yet alters the conformational flexibility of the oligonucleotide. The loss of a base also serves to disrupt the hydrogen-bonding lattice, the intramolecular base-stacking interactions, as well as the intermolecular base-stacking interactions between aromatic amino acid side chains and the substrate. Our results indicate that a gap in the charge distribution along the backbone of the substrate has a more pronounced effect upon helicase-catalyzed unwinding than does the loss of a single base. While all three substrates exhibited some degree of inhibition, analysis of both pre-steady-state and excess enzyme experiments places a greater value upon the electrostatic interactions between helicase and the substrate.
Eoff
Robert L
RL
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 West Markham Slot 516, Little Rock, Arkansas 72205, USA.
Spurling
Travis L
TL
Raney
Kevin D
KD
D017122:Bacteriophage T4 / Q000201:enzymology*; D002384:Catalysis; D004265:DNA Helicases / Q000737:chemistry* / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D004279:DNA, Viral / Q000737:chemistry* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000378:metabolism; D020135:Peptide Nucleic Acids / Q000737:chemistry / Q000378:metabolism; D055672:Static Electricity; D013379:Substrate Specificity; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D009841:Oligonucleotides; D009943:Organophosphorus Compounds; D020135:Peptide Nucleic Acids; D014764:Viral Proteins; C032627:methylphosphonic acid; D004265:DNA Helicases; C077401:dda DNA helicase protein, Bacteriophage T4