A tale of tails: insights into the coordination of 3' end processing during homologous recombination

Abstract

Eukaryotic genomes harbor a large number of homologous repeat sequences that are capable of recombining. Their potential to disrupt genome stability highlights the need to understand how homologous recombination processes are coordinated. The Saccharomyces cerevisiae Rad1-Rad10 endonuclease performs an essential role in recombination between repeated sequences, by processing 3' single-stranded intermediates formed during single-strand annealing and gene conversion events. Several recent studies have focused on factors involved in Rad1-Rad10-dependent removal of 3' nonhomologous tails during homologous recombination, including Msh2-Msh3, Slx4, and the newly identified Saw1 protein. Together, this new work provides a model for how Rad1-Rad10-dependent end processing is coordinated: Msh2-Msh3 stabilizes and prepares double-strand/single-strand junctions for Rad1-Rad10 cleavage, Saw1 recruits Rad1-Rad10 to 3' tails, and Slx4 mediates crosstalk between the DNA damage checkpoint machinery and Rad1-Rad10.

Figure 1. Single-strand annealing mechanism of double-strand break (DSB) repair between repeated sequences

DSBs that arise between repeated sequences are resected by 5′ to 3′exonuclease activity, and the single-stranded DNA is annealed at regions of homology. Pairing of these sequences reveals 3′ nonhomologous tails on either side of the intermediate that are cleaved off in a Rad1-Rad10- and Msh2-Msh3-dependent manner. Removal of the 3′ nonhomologous ends allows initiation of repair synthesis to produce final recombinant products.

Figure 2. Synthesis-dependent strand annealing mechanism of gene conversion involving the removal of either one (A) or two (B) 3′ nonhomologous tails

A. After DSB formation and 5′ to 3′ resection, one 3′ end invades a donor locus containing a homologous sequence. DNA synthesis is primed from this invading 3′ end and copies the donor sequence, and unwinding of this strand allows it to reanneal to its native locus. When the newly repaired strand differs in sequence from the original sequence, a 3′ nonhomologous tail remains at the non-invading strand. Removal of this 3′ tail involves Rad1-Rad10 and Slx4, but not Msh2-Msh3. 3′ nonhomologous tail removal allows for completion of repair by gene conversion. B. If nonhomologous sequence flanks both sides of a DSB, the 3′ invading strand must also be processed in order to have productive strand invasion. 3′ nonhomologous tail removal on the invading strand requires both the Rad1-Rad10 and Msh2-Msh3 complexes. After 3′ tail removal, gene conversion proceeds via synthesis-dependent strand annealing as described in A.

Figure 2. Synthesis-dependent strand annealing mechanism of gene conversion involving the removal of either one (A) or two (B) 3′ nonhomologous tails

A. After DSB formation and 5′ to 3′ resection, one 3′ end invades a donor locus containing a homologous sequence. DNA synthesis is primed from this invading 3′ end and copies the donor sequence, and unwinding of this strand allows it to reanneal to its native locus. When the newly repaired strand differs in sequence from the original sequence, a 3′ nonhomologous tail remains at the non-invading strand. Removal of this 3′ tail involves Rad1-Rad10 and Slx4, but not Msh2-Msh3. 3′ nonhomologous tail removal allows for completion of repair by gene conversion. B. If nonhomologous sequence flanks both sides of a DSB, the 3′ invading strand must also be processed in order to have productive strand invasion. 3′ nonhomologous tail removal on the invading strand requires both the Rad1-Rad10 and Msh2-Msh3 complexes. After 3′ tail removal, gene conversion proceeds via synthesis-dependent strand annealing as described in A.

Figure 3. Model for coordination 3′ nonhomologous tail removal factors during single-strand annealing

Following DSB formation, 5′ to 3′ resection creates single-stranded DNA that is coated by RPA. This process also signals the Mec1 and Tel1 checkpoint kinases that, once activated, phosphorylate Slx4. The Rad52 strand annealing protein facilitates pairing of the homologous sequences, displacing RPA from the annealed regions. The Msh2-Msh3 complex recognizes the junction of double-stranded and 3′ single-stranded DNA, potentially interacting with RPA, and opens the DNA junction slightly to create a better substrate for Rad1-Rad10 cleavage. The Saw1 protein recruits Rad1-Rad10 endonuclease to the junction of Rad52- and Msh2-Msh3-containing DNA, and association of Rad1-Rad10 with phosphorylated Slx4 allows it to cleave the DNA near the double-strand/single-strand junction, freeing the 3′ nonhomologous tail.