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. 1997 Dec 15;11(24):3423-31.
doi: 10.1101/gad.11.24.3423.

The preference for GT-rich DNA by the yeast Rad51 protein defines a set of universal pairing sequences

R B Tracy  1 , J K Baumohl, S C Kowalczykowski
Affiliations

The preference for GT-rich DNA by the yeast Rad51 protein defines a set of universal pairing sequences

R B Tracy et al. Genes Dev. .

Abstract

The Rad51 protein of Saccharomyces cerevisiae is a eukaryotic homolog of the RecA protein, the prototypic DNA strand-exchange protein of Escherichia coli. RAD51 gene function is required for efficient genetic recombination and for DNA double-strand break repair. Recently, we demonstrated that RecA protein has a preferential affinity for GT-rich DNA sequences-several of which exhibit enhanced RecA protein-promoted homologous pairing activity. The fundamental similarity between the RecA and Rad51 proteins suggests that Rad51 might display an analogous bias. Using in vitro selection, here we show that the yeast Rad51 protein shares the same preference for GT-rich sequences as its prokaryotic counterpart. This bias is also manifest as an increased ability of Rad51 protein to promote the invasion of supercoiled DNA by homologous GT-rich single-stranded DNA, an activity not previously described for the eukaryotic pairing protein. We propose that the preferred utilization of GT-rich sequences is a conserved feature among all homologs of RecA protein, and that GT-rich regions are loci for increased genetic exchange in both prokaryotes and eukaryotes.

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Figures

Figure 1
Figure 1
Rad51 protein selects for GT-rich DNA sequences from a random pool of oligonucleotides. In vitro selection was performed using the 54-mers containing a random internal region of 18 nucleotides. The sequences of the 18-nucleotide region after five cycles of selection and amplification are shown. In addition, the frequency of occurrence for each base is shown, along with the average base composition for all of the sequences.
Figure 2
Figure 2
Rad51 protein-promoted joint molecule formation with selected DNA. Formation of joint molecules was performed as described in Materials and Methods. (A) Joint molecule formation with pBT54CN1 and SKBT16 in the presence of either 4 mm or 10 mm magnesium acetate. Joint molecule formation was conducted with a 6-fold molar (molecule) excess of oligonucleotide SKBT16 relative to pBT54CN1; the percentage of joint molecule formation was calculated relative to the limiting amount of plasmid DNA. As a positive control, RecA protein was substituted for Rad51 protein in joint molecule formation; this reaction was conducted at 10 mm Mg2+ for 2 min. As a negative control, protein was omitted. The formation of joint molecules is indicated by the presence of 32P label migrating at the position of supercoiled pBT54CN1. The pairing reaction is depicted at the top. (B) Joint molecule formation with pBT54CN1 and either SKBT16 (▴, contains selected sequence 1); SKBT17 (•, complement of SKBT16); SKBT19 (♦, opposite side of plasmid); or SKBT20 (▪, adjacent to SKBT16). Control reactions included absence of Rad51 protein (▵); use of a heterologous plasmid (pBR322) (⋄); or absence of pBT54CN1 (□). Reactions were performed in the presence of 4 mm magnesium acetate. (C) Joint molecule formation in the presence of 10 mm magnesium acetate. In both B and C, error bars indicate the standard deviation of three experiments.
Figure 2
Figure 2
Rad51 protein-promoted joint molecule formation with selected DNA. Formation of joint molecules was performed as described in Materials and Methods. (A) Joint molecule formation with pBT54CN1 and SKBT16 in the presence of either 4 mm or 10 mm magnesium acetate. Joint molecule formation was conducted with a 6-fold molar (molecule) excess of oligonucleotide SKBT16 relative to pBT54CN1; the percentage of joint molecule formation was calculated relative to the limiting amount of plasmid DNA. As a positive control, RecA protein was substituted for Rad51 protein in joint molecule formation; this reaction was conducted at 10 mm Mg2+ for 2 min. As a negative control, protein was omitted. The formation of joint molecules is indicated by the presence of 32P label migrating at the position of supercoiled pBT54CN1. The pairing reaction is depicted at the top. (B) Joint molecule formation with pBT54CN1 and either SKBT16 (▴, contains selected sequence 1); SKBT17 (•, complement of SKBT16); SKBT19 (♦, opposite side of plasmid); or SKBT20 (▪, adjacent to SKBT16). Control reactions included absence of Rad51 protein (▵); use of a heterologous plasmid (pBR322) (⋄); or absence of pBT54CN1 (□). Reactions were performed in the presence of 4 mm magnesium acetate. (C) Joint molecule formation in the presence of 10 mm magnesium acetate. In both B and C, error bars indicate the standard deviation of three experiments.
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