Visualization of the annealing of complementary single-stranded DNA catalyzed by the herpes simplex virus type 1 ICP8 SSB/recombinase.
The rate of annealing of long linear complementary single-stranded (ss) DNAs can be increased greatly by certain DNA-binding proteins including the herpes simplex virus type 1 ICP8 SSB/recombinase. Using electron microscopy, we have investigated the DNA-protein structures involved in ICP8-mediated DNA annealing. We show that the formation of superhelical ICP8-ssDNA filaments is required for annealing. Two superhelices interact with each other to form a coiled-coil, which is the intermediate in annealing. In this process, the superhelices likely rotate and translocate relative to each other. Psoralen/UV photocrosslinking studies revealed that meta-stable contacts form at sites of limited sequence homology during the annealing. Partial proteolysis of ICP8 in the protein-ssDNA complexes showed that Mg2+ induces conformational changes in the N-terminal region (amino acid residues 1-305) of ICP8. In addition to Mg2+, Ca2+ and, to a significantly lesser extent, Cu2+ and Mn2+, were found to induce superhelix formation of the ICP8-ssDNA filament and to facilitate annealing. Mechanisms for how the coiled-coil structures facilitate annealing are discussed.
Makhov
Alexander M
AM
Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599-7295, USA. makhov@med.unc.edu
Griffith
Jack D
JD
D000818:Animals; D003432:Cross-Linking Reagents / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D004268:DNA-Binding Proteins; D005363:Ficusin / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D020673:Protein Renaturation; D045522:Recombinases / Q000378:metabolism* / Q000648:ultrastructure; D014764:Viral Proteins / Q000378:metabolism* / Q000648:ultrastructure
D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C045462:ICP8 protein, Simplexvirus; D045522:Recombinases; D014764:Viral Proteins; D008274:Magnesium; D005363:Ficusin