Abstract
The bacteriophage UvsX protein is a "strand transferase" that promotes the pairing of homologous single and double-stranded DNAs. The efficiency of UvsX protein-mediated joint molecule formation between supercoiled duplex DNA and oligonucleotides is shown to have a sharp dependence on the degree of homology. The reaction proceeded efficiently with oligonucleotides containing 32 homologous positions but not with oligonucleotides containing only 24 homologous bases. This was shown to reflect an intrinsic homology requirement for the formation of stable joint molecules and was not caused by poor binding of the protein to short single-stranded DNAs. Even a single mismatch located in the middle of a region of 40 homologous nucleotides had a detectable effect on the efficiency of pairing. An in vitro recombinationally initiated DNA synthesis reaction that mimics the "secondary mode" of phage T4 DNA replication exhibited the same homology dependence.
Highlights
The mechanism of this search for homology is unknown
It would be intrinsically interesting to know the relationship between the efficiency or rate of a homologous pairing reaction in vitro and the degree of homology shared by the DNA substrates
We show that the efficiency of joint molecule formation is poor when the DNAs share 24 or fewer homologous nucleotides but relatively high when they share 32 or more homologous positions
Summary
Vol 270, No 10, Issue of March 10, pp. 5181-5186, 1995 Printed in U.S.A. (Received for publication, January 27, 1994, and in revised form, December 18, 1994). The bacteriophage UvsX protein is a "strand transferase" that promotes the pairing ofhomologous single and double-stranded DNAs. The efficiency of UvsX proteinmediated joint molecule formation between supercoiled duplex DNA and oligonucleotides is shown to have a sharp dependence on the degree of homology. The reaction proceeded efficiently with oligonucleotides containing 32 homologous positions but not with oligonucleotides containing only 24 homologous bases This was shown to reflect an intrinsic homology requirement for the formation of stable joint molecules and was not caused by poor binding of the protein to short singlestranded DNAs. Even a single mismatch located in the middle of a region of 40 homologous nucleotides had a detectable effect on the efficiency of pairing. Once homologous regions of the DNAs have been aligned, the single-stranded DNA invades the double helix to produce a stable joint molecule, which continues down the recombination pathway The mechanism of this search for homology is unknown. A more complicated reaction in which DNA synthesis is primed by joint molecule formation exhibits the same homology dependence
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