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Papers In Press, published online ahead of print April 13, 2004
Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD 20892-0830
Corresponding Author: ngn{at}helix.nih.gov
Bacteriophage T4 gene 59 protein greatly stimulates the loading of the T4 gene 41 helicase in vitro, and is required for recombination and recombination-dependent DNA replication in vivo. 59 Protein binds preferentially to forked DNA, and interacts directly with the T4 41 helicase and gene 32 single-stranded DNA binding protein. The helicase loader is an almost completely a-helical, two domain protein, whose N-terminal domain has strong structural similarity to the DNA binding domains of high mobility group (HMG) proteins. We have previously speculated that this HMG-like region may bind the duplex ahead of the fork, with the C-terminal domain providing separate binding sites for the fork arms, and at least part of the docking area for the helicase, and 32 protein. Here, we characterize several mutants of 59 protein in an initial effort to test this model. We find that the I87A mutation, at the position where the fork arms would separate in the model, is defective in binding fork DNA. As a consequence, it is defective in stimulating both unwinding by the helicase and replication by the T4 system. 59 protein with a deletion of the two C-terminal residues, K216 and Y217, binds fork DNA normally. In contrast to the wild type, the deletion protein fails to promote binding of 32 protein on short fork DNA. However, it binds 32 protein in the absence of DNA. The deletion is also somewhat defective in stimulating unwinding of fork DNA by the helicase, and replication by the T4 system. We suggest that the absence of the two terminal residues may alter the configuration of the lagging strand fork arm on the surface of the C-domain, so that it is a poorer docking site for the helicase and 32 protein.
J. Biol. Chem, 10.1074/jbc.M402128200
Submitted on February 26, 2004
Revised on April 13, 2004
Accepted on April 13, 2004
Mutations of bacteriophage T4 59 helicase loader defective in binding fork DNA and in interactions with T4 32 single-stranded DNA binding protein
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