Aberrant double-strand break repair in rad51 mutants of Saccharomyces cerevisiae

Abstract

A number of studies of Saccharomyces cerevisiae have revealed RAD51-independent recombination events. These include spontaneous and double-strand break-induced recombination between repeated sequences, and capture of a chromosome arm by break-induced replication. Although recombination between inverted repeats is considered to be a conservative intramolecular event, the lack of requirement for RAD51 suggests that repair can also occur by a nonconservative mechanism. We propose a model for RAD51-independent recombination by one-ended strand invasion coupled to DNA synthesis, followed by single-strand annealing. The Rad1/Rad10 endonuclease is required to trim intermediates formed during single-strand annealing and thus was expected to be required for RAD51-independent events by this model. Double-strand break repair between plasmid-borne inverted repeats was less efficient in rad1 rad51 double mutants than in rad1 and rad51 strains. In addition, repair events were delayed and frequently associated with plasmid loss. Furthermore, the repair products recovered from the rad1 rad51 strain were primarily in the crossover configuration, inconsistent with conservative models for mitotic double-strand break repair.

FIG. 1

A model for RAD51-independent recombination between inverted repeats. Following introduction of a DSB in one of the repeats, one end is resected to produce a 3′ single-stranded tail, which invades the other repeat. DNA synthesis is primed from the invading end and proceeds to the end of the plasmid, coupled with lagging-strand synthesis. The sequences at the end of the linear intermediate have homology with the internal repeats. Resection and strand annealing can produce parental or inversion products. The inverted repeats are shown by thick arrows, newly synthesized DNA is indicated by dashed lines, and sequences between the repeats are designated A and B.

FIG. 2

Plasmid substrates. Plasmid pFP122 contains two copies of the lacZ gene oriented as inverted repeats. Both copies are interrupted by an insertion of 36 bp containing either the HO cut site (cs) or a point mutation to prevent HO cleavage, inc HO cut site. The plasmid contains the URA3 gene for selection in yeast and ARS and CEN elements for stable replication as an episome. After induction of HO, repair can occur by gene conversion to form noncrossover or crossover products, which can be distinguished by restriction digestion with PstI (P). Failure to repair results in plasmid loss and a Ura⁻ phenotype. Plasmid pJFL33 contains direct repeats of lacZ interrupted by an insertion of 117 bp with the HO or inc HO cut site, URA3 and LEU2 genes, and ARS and CEN elements for stable maintenance. Restriction sites for BclI (B), HindIII (H), PstI (P), and XhoI (X) are shown.

FIG. 3

Delayed plasmid repair and plasmid loss in rad51 mutants. (A) Strains containing the inverted-repeat plasmid were plated onto solid medium containing either glucose or galactose, and colony size was analyzed after 3 or 4 days of growth. (B) Colonies were replica plated to SC−Ura after 4 to 5 days of growth on YEP-galactose plates to measure plasmid retention and colony sectoring.

FIG. 4

Repair is delayed in rad51 mutants. (A) Substrate and products detected by PstI (P) digestion. (B) Kinetic analysis of repair in each strain. Samples were removed prior to HO induction (0 h) and at hourly intervals after HO induction. DNA samples were digested with PstI and fragments were separated on 0.6% agarose gels prior to transfer. Sizes are indicated in kilobases. (C) Hybridized filters were analyzed using a Molecular Dynamics PhosphorImager, and the recombination products were normalized to unique chromosomal sequences to account for plasmid loss. □, wild type; ◊, rad1 mutant; ○, rad51 mutant; ▵, rad1 rad51 double mutant.

FIG. 5

Kinetic analysis of repair of plasmid pJFL33. (A) pJFL33 contains direct repeats of lacZ interrupted by a 117-bp insertion of the HO cut site (cs) or inc HO cut site. Repair of the HO-induced DSB can occur by SSA to delete one copy of lacZ, by gene conversion unassociated with crossing over, or by formation of a triplication of lacZ. Each of these products can be distinguished by restriction digestion with PstI (P), HindIII (H), and BclI (B). HO cleavage produces a fragment of 1.45 kb, the SSA product produces a 6.8-kb PstI fragment, and the triplication product generates a 4-kb HindIII fragment. The conversion product produces PstI/HindIII fragments of the same size as the parental plasmid but can be monitored by the appearance of 1.4- and 1.5-kb BclI/HindIII fragments generated by conversion of restriction site polymorphisms flanking the HO cut site. X, XhoI. (B) DNA samples were digested with PstI and HindIII to detect the deletion and triplication products. Sizes are indicated in kilobases. (C) The percentages of total plasmid DNA represented by the HO cut fragments, deletion products, and triplication products were quantitated by phosphorimaging and shown graphically. (D) DNA samples were digested with PstI, HindIII, and BclI to detect the conversion products. Sizes are indicated in kilobases. (E) Quantitation of the 1.5-kb conversion product shown in panel D. □, wild type; ◊, rad1 mutant; ○, rad51 mutant; ▵, rad1 rad51 double mutant.

FIG. 6

Model for BIR/SSA within the direct-repeat plasmid (12). One-ended invasion of the unbroken lacZ gene to prime DNA synthesis to the end of the linear molecule results in duplication of the lacZ gene. Depending on how the complementary sequences pair, a deletion or apparent conversion can result. If the upstream (U) sequence of the broken repeat pairs with the upstream (U′) sequence of the unbroken repeat, and the newly synthesized downstream (D′) sequences pair with the broken repeat (D), then DNA synthesis could initiate from the other side of the break to create an additional repeat. Dissociation and realignment are required to generate the triplication product. The deletion and conversion products require clipping of a 3′ heterologous tail, whereas the triplication product has no tails to be removed and thus is favored in rad1 strains.

FIG. 7

Plasmid recovery through replication bypass of the Rad1 trimming step. The annealed intermediate formed by SSA when the HO cut site is within heterologous sequences is predicted to have two 3′ heterologous tails flanking the annealed region. This molecule is predicted to form inviable products if replicated. When SSA occurs between repeats that are completely homologous, only one 3′ heterologous tail is predicted to form. If the other strand is ligated and the plasmid undergoes replication, one viable daughter should be generated.

FIG. 8

Intermolecular recombination to produce an inversion product. If two plasmids pair in an antiparallel configuration, BIR initiated from one cut repeat, coupled with lagging-strand synthesis, will duplicate the intervening sequences to form an inversion. DNA synthesis will be blocked at the break in the second plasmid, leading to strand displacement, annealing, and removal of the 3′ heterologous tail, resulting in formation of an inversion product.