Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 355, Issue 5, Pages 911-922Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2005.11.022
Keywords
ICP8; herpes simplex virus; SSB; electron microscopy; single-stranded DNA annealing
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Funding
- NCI NIH HHS [CA 19014] Funding Source: Medline
- NIGMS NIH HHS [GM31819] Funding Source: Medline
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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 roteolysis 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. (c) 2005 Elsevier Ltd. All rights reserved.
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