Cloning of the bacteriophage T4 uvsX gene and purification and characterization of the T4 uvsX recombination protein.

D M Hinton,N G Nossal

doi:10.1016/s0021-9258(19)57266-0

Abstract

The bacteriophage T4 uvsX gene is a nonessential gene required for normal levels of DNA repair, recombination, and replication. We demonstrate that plasmids containing the T4 DNA approximately 300-2900 base pairs upstream of T4 gene 41 express a biologically active uvsX protein. This uvsX protein imparts increased survival to UV-irradiated T4 uvsX- phage and decreases the T4 uvsX- mutant suppression of a conditionally lethal T4 mutant in the gene 49 recombination nuclease. The uvsX protein purified from cells with a uvsX+ plasmid catalyzes ATP hydrolysis to ADP and AMP and, in the presence of the T4 gene 32 helix-destablizing protein, ATP-dependent strand exchange between homologous circular single-stranded and linear duplex DNA. These results agree with the recent characterization of uvsX protein from T4-infected cells by Yonesaki et al. (Yonesaki, T., Ryo, Y., Minagawa, T., and Takahashi, H. (1985) Eur. J. Biochem. 148, 127-134) and by Formosa and Alberts (Formosa, T., and Alberts, B.M. (1984) Cold Spring Harbor Symp. Quant. Biol. 49, 363-370). In addition, we find that under some reaction conditions strand exchange is catalyzed by uvsX protein in the absence of 32 protein. The level of the uvsX protein expressed by the uvsX+ plasmids is high and independent of the orientation of the T4 DNA within the vector. This suggests that transcription promoter(s) lie upstream of the uvsX gene on the cloned T4 DNA. In vitro transcription of T4 DNA restriction fragments reveals two tandem promoters whose transcripts initiate approximately 500 and 600 nucleotides upstream of the uvsX gene and extend through the gene.

Highlights

DNA, These results agree with the recent characteri- extended by the complex of replication enzymes
Genetic mapping experiments haveassigned the UUSXgene upstream of gene 41 [22,30].We have located gene 41 on the physical map of T4 by showing that plasmids containing T4 map units22.01 to 20.06 express 41 protein [31].In theprocess of constructing plasmids containing gene 41, we have found that theregion upstream of gene 41 encodes a protein of about 46 kDa. We show that this proteinis the T4uvsX gene product and that the presence of a plasmid expressing uvsX protein in the cell complements T4 U U S X -phage
We find no P-glucosyltransferase expressed by our clones behaves like a biologically active activity incrude extracts of uvsX gene product

Summary

RESULTS

On the translationproducts expressed by the plasmid pDH428A3, a plasmid identical to pDH428 except that the. Abnormal 30-kDa protein by pDH428A3 indicates that the normal gene starts upstream of the CZaI site at map unit 22.85 and proceeds toward gene 41 This direction is consistentwith the previously determined general direction of early T4 transcription in vivo [2], the direction of the immediately down-. The 46-kDa protein expressed by this gel is nondenaturing the sizes for the RNAs cannot be pDH447 binds to the dsDNA cellulose in the presence of 0.1 determined, the major species from transcription of M NaCl, is eluted by stepping the saltconcentration to 0.5 M the EcoRI fragment appears tobe shorter than thaot btained NaCl (lune E), andco-migrates with a bound protein after transcriptionof the SalItemplate.

VECTOR

DISCUSSION

EXPERIMENTAL PROCEDURES