The preference for GT-rich DNA by the yeast Rad51 protein defines a set of universal pairing sequences - PubMed (original) (raw)

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 m

m

or 10 m

m

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 m

m

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 m

m

magnesium acetate. (C) Joint molecule formation in the presence of 10 m

m

magnesium acetate. In both B and C, error bars indicate the standard deviation of three experiments.