Kinetic regulation of single DNA molecule denaturation by T4 gene 32 protein structural domains.
Bacteriophage T4 gene 32 protein (gp32) specifically binds single-stranded DNA, a property essential for its role in DNA replication, recombination, and repair. Although on a thermodynamic basis, single-stranded DNA binding proteins should lower the thermal melting temperature of double-stranded DNA (dsDNA), gp32 does not. Using single molecule force spectroscopy, we show for the first time that gp32 is capable of slowly destabilizing natural dsDNA. Direct measurements of single DNA molecule denaturation and renaturation kinetics in the presence of gp32 and its proteolytic fragments reveal three types of kinetic behavior, attributable to specific protein structural domains, which regulate gp32's helix-destabilizing capabilities. Whereas the full-length protein exhibits very slow denaturation kinetics, a truncate lacking the acidic C-domain exhibits much faster kinetics. This may reflect a steric blockage of the DNA binding site and/or a conformational change associated with this domain. Additional removal of the N-domain, which is needed for binding cooperativity, further increases the DNA denaturation rate, suggesting that both of these domains are critical to the regulation of gp32's helix-destabilization capabilities. This regulation is potentially biologically significant because uncontrolled helix-destabilization would be lethal to the cell. We also obtain equilibrium measurements of the helix-coil transition free energy in the presence of these proteins for the first time.
Pant
Kiran
K
Department of Physics, Northeastern University, 111 Dana Research Center, Boston, MA 02115, USA.
Karpel
Richard L
RL
Williams
Mark C
MC
D017122:Bacteriophage T4 / Q000378:metabolism; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004261:DNA Replication; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D018715:Microscopy, Video; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013053:Spectrophotometry; D013696:Temperature; D013997:Time Factors; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C060035:gp32 protein, Enterobacteria phage T4; D004247:DNA