The DNA damage checkpoint allows recombination between divergent DNA sequences in budding yeast

Abstract

In the early steps of homologous recombination, single-stranded DNA (ssDNA) from a broken chromosome invades homologous sequence located in a sister or homolog donor. In genomes that contain numerous repetitive DNA elements or gene paralogs, recombination can potentially occur between non-allelic/divergent (homeologous) sequences that share sequence identity. Such recombination events can lead to lethal chromosomal deletions or rearrangements. However, homeologous recombination events can be suppressed through rejection mechanisms that involve recognition of DNA mismatches in heteroduplex DNA by mismatch repair factors, followed by active unwinding of the heteroduplex DNA by helicases. Because factors required for heteroduplex rejection are hypothesized to be targets and/or effectors of the DNA damage response (DDR), a cell cycle control mechanism that ensures timely and efficient repair, we tested whether the DDR, and more specifically, the RAD9 gene, had a role in regulating rejection. We performed these studies using a DNA repair assay that measures repair by single-strand annealing (SSA) of a double-strand break (DSB) using homeologous DNA templates. We found that repair of homeologous DNA sequences, but not identical sequences, induced a RAD9-dependent cell cycle delay in the G2 stage of the cell cycle. Repair through a divergent DNA template occurred more frequently in RAD9 compared to rad9Δ strains. However, repair in rad9Δ mutants could be restored to wild-type levels if a G2 delay was induced by nocodazole. These results suggest that cell cycle arrest induced by the Rad9-dependent DDR allows repair between divergent DNA sequences despite the potential for creating deleterious genome rearrangements, and illustrates the importance of additional cellular mechanisms that act to suppress recombination between divergent DNA sequences.

Introduction

The DNA damage response (DDR) plays a central role in ensuring that critical biological processes, such as immunoglobulin diversification, gamete development, and telomere homeostasis, occur with limited errors [1], [2], [3]. These processes rely on programmed genomic insults that are repaired in a highly regulated manner, and the DDR is essential for coordinating their repair with cell growth and division. Humans and mice with defects in the DDR exhibit increased genomic instability and can display increased incidence of cancer, neurodegeneration, immunodeficiency, or infertility (i.e. Ataxia telangiectasia, Nijmegen breakage syndrome, Down's syndrome, Alzheimer's disease [4]).

Genome stability is maintained by groups of proteins that recognize and repair DNA damage in the form of replication or recombination errors and chemically or radioactively induced lesions [4], [5], [6], [7], [8]. DNA double-strand breaks (DSBs) are cytotoxic type of DNA damage that can result from strand breakage associated with physical stress, ionizing radiation, endonuclease cleavage, stalled intermediates in DNA lesion processing, and replication fork collapse [9], [10], [11], [12], [13], [14], [15], [16]. DSBs are often repaired by one of several forms of chromosomal recombination, and their timely and accurate repair is essential for avoiding the genomic rearrangements that can lead to disease.

In budding yeast the DDR is critical for promoting efficient repair of DSBs. Upon formation of a DSB, a single DNA strand is resected from each broken end in the 5′–3′ direction, exposing 3′ single-stranded DNA (ssDNA). The ssDNA is immediately bound by RPA, followed by binding of complexes containing the Mec1/Tel1 PIKK protein kinases and Rad9 [17], [18]. Rad9 is phosphorylated by Mec1/Tel1 and forms an oligomer which serves as a scaffold for Rad53, allowing for Rad53 autophosphorylation [19]. Rad53 is the central DDR transducer which signals to many downstream effectors to promote localization of DNA repair factors to sites of damage and delays cell cycle progression to ensure that the damage is repaired before cell division [19], [20], [21]. If a DSB fails to be repaired, the cell will either remain terminally arrested at the G2/M stage of the cell cycle or will undergo break adaptation and die after several divisions [22], [23], [24].

Though the DDR has been widely studied, our understanding of all of its downstream steps is far from complete. One area that is not well understood is the role of the DDR in the choice of DSB repair pathway and the recognition of the correct repair template for homologous recombination. DSB repair may occur by the non-conservative non-homologous end joining pathway (NHEJ), or by one form of conservative homologous recombination (HR) including classical double-strand break repair (DSBR), synthesis-dependent strand annealing (SDSA), single-strand annealing (SSA), or break-induced replication (BIR), all of which initiate with strand invasion or annealing of homologous DNA sequences which are then used as templates for DNA synthesis to fill in sequence gaps [25]. The current understanding is that the DSBR and SDSA pathways are preferred during the late S or G2 stage of the cell cycle or meiotic pachytene when chromosomes are in close proximity to a sister chromatid or homologous chromosome. In contrast, NHEJ is functional at all cell cycle stages, and therefore is primarily responsible for repairing breaks during the G0 or G1 stages in mammals (though it plays a smaller role in DSB repair in yeast) when sister chromatids are unavailable [26], [27]. Finally, SSA and BIR are specialized for repair of DSBs within repetitive DNA elements and when only one DSB end has a homologous template, respectively [28].

Little is known about how the DDR affects the choice of homologous repair template during HR. Despite the fact that approximately 50% of the human genome is composed of repetitive DNA elements [29], homologous template choice for DSB repair is still chosen with remarkable fidelity [30]. Still, a number of genome rearrangements between repetitive elements have been described that have been associated with cancers and neurological diseases, including familial breast and ovarian cancer as well as Charcot-Marie-Tooth disease [31], [32], [33], [34], highlighting the need to further understand the mechanisms that control HR. Some of the factors required for homologous template choice have been identified [35], [36], [37], [38], [39], [40], [41], [42]; however, the role of the DDR in this choice has not been explored. Here, we tested a role of the DDR in homologous partner choice using SSA between direct DNA repeats as a model. Previous work using this model [35] has shown that SSA between 205 bp repetitive elements spaced 2.6 kb apart is repaired efficiently by annealing of complementary DNA on resected ssDNA ends, cleavage of the 3′ tails derived from the intervening non-homologous sequences, and filling of gaps by DNA synthesis and ligation to create a double-stranded DNA (dsDNA) deletion product (Fig. 1). However, SSA repair at the same locus is inefficient when the repetitive elements share less-than-perfect sequence identity, except when factors critical for disrupting the heteroduplex intermediate (Msh6 or Sgs1) are absent [35]. The process for disruption of divergent SSA intermediates, termed heteroduplex rejection, occurs by a conservative unwinding mechanism such that rejected intermediates still have the potential to repair correctly if the appropriate homologous template is available [36].

To determine whether the RAD9-dependent DDR is involved in the formation or rejection of heteroduplex SSA intermediates, we compared the effectiveness of heteroduplex rejection in a wild-type versus rad9Δ strain background. Unexpectedly, we found that heteroduplex rejection was less efficient in the presence of the DDR than in its absence; DDR allowed recombination between divergent sequences. Further analysis showed that a G2 delay occurred in wild-type strains that allowed divergent recombination, and inducing a synthetic G2 delay in rad9Δ mutants by adding nocodazole was able to restore the wild-type level of rejection. These results are the first to show a role for the DDR in allowing inappropriate error-prone DSB repair over error-free repair. This work also provides insights into how repetitive DNA can threaten the integrity of the genome and suggests a new explanation for why some disease-causing rearrangements could escape mechanisms that normally suppress them.