Risky business: Microhomology-mediated end joining

Abstract

Prevalence of microhomology (MH) at the breakpoint junctions in somatic and germ-line chromosomal rearrangements and in the programmed immune receptor rearrangements from cells deficient in classical end joining reveals an enigmatic process called MH-mediated end joining (MMEJ). MMEJ repairs DNA double strand breaks (DSBs) by annealing flanking MH and deleting genetic information at the repair junctions from yeast to humans. Being genetically distinct from canonical DNA DSB pathways, MMEJ is involved with the fusions of eroded/uncapped telomeres as well as with the assembly of chromosome fragments in chromothripsis. In this review article, we will discuss an up-to-date model representing the MMEJ process and the mechanism by which cells regulate MMEJ to limit repair-associated mutagenesis. We will also describe the possible therapeutic gains resulting from the inhibition of MMEJ in recombination deficient cancers. Lastly, we will embark on two contentious issues associated with MMEJ such as the significance of MH at the repair junction to be the hallmark of MMEJ and the relationship of MMEJ to other mechanistically related DSB repair pathways.

Keywords: Chromosomal rearrangements; DNA double strand break repair; Microhomology.

Fig. 1. Basic MMEJ mechanism

NHEJ preferentially repairs DNA breaks with limited or no resection. Binding of Ku impedes resection and 0–3 bp MHs (pink boxes) helps juxtaposing DNA ends, yielding repair products with 1–5 bp deletions/insertions at the repair junctions. Alternatively, DNA resection exposes 2–20 bp (MMEJ) or >15 bp (SSA) homology (pink boxes) at the flanking sequence for annealing DNA ends in MMEJ and SSA. In both MMEJ and SSA, homology annealing is followed by 3′ flap trimming, DNA synthesis and ligation, producing MMEJ products with various size of deletions/insertions or SSA products with large deletions but no inserted nucleotides.

Fig. 2. Synthesis dependent MMEJ

MMEJ could occur between MHs (z) that forms by repair synthesis of the same chromosome (A) or other chromosome (B) after annealing of DNA ends using limited homology (shown in x). The broken end (Ai) is resected (Aii), forms a loop after unwinding and uses nearby sequences as template to synthesize DNA (Aiii). After synthesis, the loop dissociates or unwinds (Aiv) to generate a 3′ overhang that has microhomology (z) complementary to the break for annealing (Av). The resulting repair product formed due to MMEJ leads net insertion at the breakpoint junction (Avi). Alternatively, the broken end (black chromosome) switches template(grey chromosome) using microhomology (Bi). Sequence from another genomic locus is copied (Bii) to acquire microhomology (z), which is used to anneal after switching back to the original template (Biii). Such repair products do not have flanking MHs at the breakpoint junctions as predicted but may carry insertions or duplications of the sequence (y) adjacent to newly copied MHs.

Fig. 3. Microhomology-mediated break induced replication (mmBIR) and Fork Stalling/Template Switching (FoSTeS)

A. Microhomology-mediated break-induced replication (MMBIR). (i) The ongoing replication fork may encounter a nick or lesion on the template strand, which leads to replication fork collapse. (ii) The replication fork is then collapsed forms a double stranded break. The 5′ end of the broken end undergoes resection, exposing a 3′ tail. (iii) After resection, the 3′end invades different template (grey) using microhomology (mh1). Replication is re-initiated but of low processivity. (iv and v) The extended broken end, now carrying the different sequence (green) dissociates and reinvades different templates (pink) using another microhomology (mh2). (v and vi) This process of template switching continues until the extended end anneals back with the original single-stranded sequence (black) using microhomology (mh3). The fully replication fork is re-formed and the synthesis resumes till the end of the replicon. B. Fork Stalling and Template Switching (FoSTeS)​ (i) Replication fork stalling can be caused by the formation of secondary structures, lesions or shortage of deoxynucleotide triphosphates in lagging strand template. (ii) The 3′ primer end of the DNA strand becomes dissociated from their templates and might align to single-stranded DNA templates in other nearby replication forks (grey) that share microhomology (mh1). (iii) This process could occur multiple times by repetitive dissociation and reinvasion into nearby replication forks. (iv) According to the position of the other replication fork, the resolution of this intermediate might lead to the formation of chromosomal rearrangements like duplication, deletion or translocation.