A mechanistic basis for Mre11-directed DNA joining at microhomologies

Abstract

Repair of DNA double-strand breaks in vertebrate cells occurs mainly by an end-joining process that often generates junctions with sequence homologies of a few nucleotides. Mre11 is critical for this mode of repair in budding yeast and has been implicated in the microhomology-based joining. Here, we show that Mre11 exonuclease activity is sensitive to the presence of heterologous DNA, and to the structure and sequence of its ends. Addition of mismatched DNA ends stimulates degradation of DNA by Mre11, whereas cohesive ends strongly inhibit it. Furthermore, if a sequence identity is revealed during the course of degradation, it causes Mre11 nuclease activity to pause, thus stabilizing the junction at a site of microhomology. A nuclease-deficient Mre11 mutant that still binds DNA can also stimulate degradation by wild-type Mre11, suggesting that Mre11-DNA complexes may interact to bridge DNA ends and facilitate DNA joining.

Figure 1

Heterologous DNA molecules increase Mre11 exonuclease activity. A total of 25 ng (0.3 pmol) of human recombinant Mre11 was incubated with 0.04 pmol of double-stranded DNA containing a 4-nt 5′ overhang, labeled at the 5′ end of the recessed strand. Immediately after combining Mre11 with the labeled substrate, various unlabeled DNA molecules were added, as indicated. The fold excess of competitor was calculated by using moles of DNA molecules. The reactions were stopped after 30 min and then run on a denaturing polyacrylamide gel.

Figure 2

The end sequence of heterologous DNA molecules determines the effect on Mre11 exonuclease activity. (A) 0.3 pmol Mre11 was incubated with 0.04 pmol of double-stranded DNA containing a 4-nt 5′ overhang and labeled at the 5′ end of the recessed strand, as shown. Various unlabeled DNAs were added, as in Fig. 1. The arrows indicate positions in the substrate DNA that correspond to the 5′ edge of sequence homologies present in both the substrate and the competitor. The presence of the specific competitor DNAs induces an increase in accumulation of the band indicated because of microhomology-induced pausing of Mre11 exonuclease activity (see model in Fig. 5). (B) 0.3 pmol Mre11 was incubated with 0.04 pmol of double-stranded DNA containing an 11-nt 5′ overhang and labeled at the 5′ end of the recessed strand, as shown. Various unlabeled DNAs containing 5′ overhangs were added as in Fig. 1, except that 10-fold lower amounts were used.

Figure 3

A nuclease-deficient but DNA-binding-proficient Mre11 mutant increases the activity of wild-type Mre11. (A) Gel mobility shift assay: 200 ng of wild-type or H217Y Mre11, as indicated, were incubated with a labeled double-stranded DNA containing 3′ overhangs on each end and run on a 6% acrylamide gel. (B) 30 ng of wild-type Mre11 was incubated with the same 5′ overhang substrate as in Fig. 2B. Increasing amounts (30 ng, lanes 3 and 6; 60 ng, lanes 4 and 7; 120 ng, lanes 5 and 8) of H217Y Mre11 protein were included in the reactions as indicated.

Figure 4

Wild-type and H217Y Mre11 increase joining of nonhomologous and homologous ends. Plasmid DNA was cut with restriction enzymes to generate mismatched overhangs (XhoI/BamHI), cohesive 5′ overhangs (BamHI/BglII), or cohesive 3′ overhangs (SphI). The plasmids were incubated with varying amounts of wild-type or H217Y Mre11, as well as with ligase IV/Xrcc4 complex, as indicated. The products were amplified by PCR and run on a native polyacrylamide gel. Arrows indicate the positions of the uncut plasmid DNA and the joining products.

Figure 5

A hypothetical model of microhomology searching by Mre11. (A) Broken DNA ends are generated that contain a sequence identity (“ATG”) in the single-stranded overhang of one end (thick lines) that is also present in the double-stranded region of the other end (thin lines). (B) A putative multimer of Mre11 binds both DNA ends simultaneously. The 5′ overhangs do not match, thus Mre11 exonuclease activity is stimulated on one end (here shown as the end on the right). (C) After excision of a few nucleotides, the overhangs still cannot base pair with one another, so Mre11 exonuclease activity proceeds further. (D) Degradation of the ATG present on the bottom strand of the right-hand DNA molecule uncovers the TAC on the top strand, thus creating cohesive ends and pausing exonuclease activity, which increases the probability of a DNA ligase sealing the nick on the bottom strand. Other enzymes would then be necessary to repair the top strand.