Single-strand annealing between inverted DNA repeats: Pathway choice, participating proteins, and genome destabilizing consequences

Abstract

Double strand DNA breaks (DSBs) are dangerous events that can result from various causes including environmental assaults or the collapse of DNA replication. While the efficient and precise repair of DSBs is essential for cell survival, faulty repair can lead to genetic instability, making the choice of DSB repair an important step. Here we report that inverted DNA repeats (IRs) placed near a DSB can channel its repair from an accurate pathway that leads to gene conversion to instead a break-induced replication (BIR) pathway that leads to genetic instabilities. The effect of IRs is explained by their ability to form unusual DNA structures when present in ssDNA that is formed by DSB resection. We demonstrate that IRs can form two types of unusual DNA structures, and the choice between these structures depends on the length of the spacer separating IRs. In particular, IRs separated by a long (1-kb) spacer are predominantly involved in inter-molecular single-strand annealing (SSA) leading to the formation of inverted dimers; IRs separated by a short (12-bp) spacer participate in intra-molecular SSA, leading to the formation of fold-back (FB) structures. Both of these structures interfere with an accurate DSB repair by gene conversion and channel DSB repair into BIR, which promotes genomic destabilization. We also report that different protein complexes participate in the processing of FBs containing short (12-bp) versus long (1-kb) ssDNA loops. Specifically, FBs with short loops are processed by the MRX-Sae2 complex, whereas the Rad1-Rad10 complex is responsible for the processing of long loops. Overall, our studies uncover the mechanisms of genomic destabilization resulting from re-routing DSB repair into unusual pathways by IRs. Given the high abundance of IRs in the human genome, our findings may contribute to the understanding of IR-mediated genomic destabilization associated with human disease.

Genome plasticity in Candida albicans is driven by long repeat sequences

“…We next calculated the orientation and distance between matched intra-chromosomal repeat sequences (Figure 2—figure supplement 1), both important factors for reconstructing the evolutionary history of these duplication events and for analyzing the frequency and outcome of homologous recombination events that occur between repeat sequences (Lobachev et al, 1998; Ramakrishnan et al, 2018). Intra-chromosomal repeats are often generated in tandem by recombination between sister chromatids or replication slippage, and these repeats can move further away from each other by chromosomal rearrangement events (including chromosomal inversions) (Achaz et al, 2000; Reams and Roth, 2015).…”

“…First, we propose that single-strand annealing (SSA) is initiated by the annealing of DNA repeats that become single-stranded after a DSB and 5’−3’ DNA resection (Figure 7A–7B) and occurs between both tandem and inverted repeat sequences (Bhargava et al, 2016; Malkova and Haber, 2012; Mehta and Haber, 2014; Ramakrishnan et al, 2018; VanHulle et al, 2007). SSA that occurs between tandem repeats leads to segmental deletion of the sequence located between the repeat sequences (Figure 7C).…”

Expandable and reversible copy number amplification drives rapid adaptation to antifungal drugs

“…This DSB generates a telomere proximal acentric fragment that is lost during future cell divisions. After DNA replication, NAHR between the long inverted repeat sequences ( Figure 6A and B , Repeat 124, blue arrows) located on sister chromatids generates a dicentric chromosome and amplification of all sequence from the site of NAHR to the telomere on Chr3R ( Croll et al, 2013 ; Lang et al, 2013 ; Ramakrishnan et al, 2018 ; Stimpson et al, 2012 ). Alternatively, the dicentric chromosome could be formed via an intra-chromosomal fold-back-like mechanism between repeat copies that primes break induced replication (BIR) ( Narayanan et al, 2006 ; Rattray et al, 2005 ).…”

Centromeres under Pressure: Evolutionary Innovation in Conflict with Conserved Function

“…As a result, DNA repair protein RAD51 homolog 1 (RAD51) plays a central role in processing the HJ loop [ 194 ], principally with the aim to inhibit single-strand annealing (SSA), an error-prone mechanism that anneals the homologous DNA sequence at the break without a gap, causing a sequence deletion ( Figure 3 F) [ 187 , 195 ]. SSA results in loss of DNA where the 25-nucleotide strand annealing is followed only by polymerase filling and intermediate ligation [ 196 , 197 ]. Because many of these repair pathways are error-prone, they induce mutagenesis that may favor the evolution of centromere DNA ( Figure 3 ).…”