Rad52 and Ku bind to different DNA structures produced early in double-strand break repair

Abstract

DNA double-strand breaks are repaired by one of two main pathways, non-homologous end joining or homologous recombination. A competition for binding to DNA ends by Ku and Rad52, proteins required for non-homologous end joining and homologous recombination, respectively, has been proposed to determine the choice of repair pathway. In order to test this idea directly, we compared Ku and human Rad52 binding to different DNA substrates. How ever, we found no evidence that these proteins would compete for binding to the same broken DNA ends. Ku bound preferentially to DNA with free ends. Under the same conditions, Rad52 did not bind preferentially to DNA ends. Using a series of defined substrates we showed that it is single-stranded DNA and not DNA ends that were preferentially bound by Rad52. In addition, Rad52 aggregated DNA, bringing different single-stranded DNAs in close proximity. This activity was independent of the presence of DNA ends and of the ability of the single-stranded sequences to form extensive base pairs. Based on these DNA binding characteristics it is unlikely that Rad52 and Ku compete as 'gatekeepers' of different DNA double-strand break repair pathways. Rather, they interact with different DNA substrates produced early in DNA double-strand break repair.

Figure 1

Schematic representation of the steps in synthesis of the DNA substrate with an internal single-stranded gap. The 810 bp PCR I DNA, shown in black at the left, formed the basis for building up the complete product. It was first digested with lambda exonuclease III to produce the bottom strand, to which oligo DR1, red, was hybridized and used as a primer for synthesis of double-stranded DNA, blue, toward one end. The 313 bp PCR II DNA, shown in green at the right, was identical in sequence to the first 313 bp of PCR I but has a 5′ phosphate on the opposite strand as PCR I. Digestion of PCR II with lambda exonuclease left a 313 nt single-stranded DNA complementary to the PCR I bottom strand. Upon annealing, this resulted in the final product with blunt ends and an internal 200 nt single-stranded gap.

Figure 2

Diagram of the DNA substrates used in this study. The length as well as type of ends for each DNA is indicated. All but the substrate with the internal 213 nt gap were derived from 1.8 kb plasmid pDERI1. The substrate with the internal 213 nt gap was derived from the S.cerevisiae URA3 gene.

Figure 3

Human Rad52 forms large oligomers on long single-stranded DNA. (A) SFM images showing large Rad52 complexes formed in binding reactions including DNA with long single-stranded ends. The long single-stranded DNA ends not bound by protein appear as a small knob at the end of the substrate. (B) SFM images showing small Rad52 complexes formed in binding reactions including DNA with blunt ends (left), short 5′ overhang ends (middle) and short 3′ overhang ends (right). The scale bar indicates 300 nm in the X and Y dimensions and height is represented by color as shown by the bar at the right.

Figure 4

Competition for binding to a mixture of DNA substrates by human Rad52 or Ku. (A) SFM images from binding reactions including Rad52 and a mixture of 1.8 kb nicked circular DNA, 1.8 kb linear blunt-ended DNA and 0.9 kb DNA with a 200 nt single-stranded end. Rad52 bound exclusively to the long single-stranded DNA as large oligomers. (B) SFM images from binding reactions including Ku and the same mixture of DNA substrates as in (A). Ku bound almost exclusively to the linear DNA substrates independent of the end structure. The scale bar indicates 200 nm in the X and Y dimensions and height is represented by color as shown by the bar at the right.

Figure 5

Human Rad52 complexes often aggregate single-stranded DNA. (A) SFM images from binding reactions including Rad52 and DNA with one long single-stranded end. Large Rad52 oligomers form on the single-stranded end of the DNA and often aggregate more than one DNA molecule. (B) SFM images from binding reactions including Rad52 and DNA with a central single-stranded gap. Large Rad52 oligomers form on the central single-stranded region of this DNA and often aggregate more than one DNA molecule. The scale bar indicates 300 nm in the X and Y dimensions, height ranges from 0 to 4 nm and is represented by color (red to yellow as in the scale bars of Figs 3 and 4).