DNA double-strand break repair-pathway choice in somatic mammalian cells

Abstract

The major pathways of DNA double-strand break (DSB) repair are crucial for maintaining genomic stability. However, if deployed in an inappropriate cellular context, these same repair functions can mediate chromosome rearrangements that underlie various human diseases, ranging from developmental disorders to cancer. The two major mechanisms of DSB repair in mammalian cells are non-homologous end joining (NHEJ) and homologous recombination. In this Review, we consider DSB repair-pathway choice in somatic mammalian cells as a series of 'decision trees', and explore how defective pathway choice can lead to genomic instability. Stalled, collapsed or broken DNA replication forks present a distinctive challenge to the DSB repair system. Emerging evidence suggests that the 'rules' governing repair-pathway choice at stalled replication forks differ from those at replication-independent DSBs.

Figure 1.. Two major pathways of DNA double strand break repair.

The binding of the Ku70–Ku80 heterodimer to DNA ends schedules repair of DNA double strand breaks (DSBs) by classical non-homologous end joining (cNHEJ). cNHEJ entails formation of a ‘long range’ synaptic complex, which is in equilibrium with a ‘short range’ synaptic complex. End processing by cNHEJ enzymes (as shown) and ligation are restricted to the short range complex. PNKP: Polynucleotide kinase-phosphatase. TDP1: Tyrosyl-DNA phosphodiesterase 1. The default engagement of cNHEJ can be disrupted by DNA end resection. The nuclease activity of MRE11 converts the blunt end into a 3ʹ single-stranded DNA (ssDNA) tail, displacing Ku70–Ku80 from the DNA end and establishing the possibility of repair by homologous recombination (HR). The replication protein A (RPA) complex avidly binds to ssDNA and must be displaced by recombination mediators to enable the formation of a RAD51 nucleoprotein filament. BRCA2 is the major recombination mediator in mammalian cells, likely acting in concert with PALB2 and the BRCA1–BARD1 heterodimer. Interactions between the two DNA ends at the recombination synapse, and operations on the D-loop formed following synapsis, influence which HR sub-pathway is engaged. The non-crossover synthesis-dependent strand annealing (SDSA) pathway is the predominant repair pathway in somatic cells. In meiotic cells, formation of a double Holliday junction (dHJ) intermediate can lead to crossing over. A failure to engage the second end of the break, or failure to displace the nascent strand leads to aberrant replicative HR responses of long tract gene conversion (LTGC) and break-induced replication (BIR). Established roles for BRCA gene products in HR are indicated in parentheses.

Figure 2.. Alternative DSB repair pathways.

A. Single strand annealing (SSA) converts homologous repeats (marked in green) to a single copy, by annealing complementary single-stranded DNA (ssDNA) ends within each repeat. Replication protein A (RPA) must be displaced to expose complementary ssDNA for annealing. B. Alternative end joining (aEJ) rejoins DNA ends without use of classical non-homologous end joining (cNHEJ) proteins. MH: microhomology. Frequent use of microhomology-mediated end joining (MMEJ) is typical but not a defining feature of aEJ. The figure depicts the action of DNA polymerase θ (Pol θ), an important aEJ mediator in mammalian cells. C. Microhomology-mediated template switching can arise when a free 3ʹ ssDNA end lacks an immediately available partner for recombination or rejoining. The persistent ssDNA end is thought to interact with ssDNA gaps in neighbouring DNA molecules, leading to the synthesis of a few hundred base pairs templated on the ectopic donor strand. Multiple rounds of microhomology-mediated or HR-mediated template switching can give rise to complex breakpoints in cancer and in developmental disorders.

Figure 3.. A decision tree of DNA double strand break repair.

DNA end resection has a crucial role in determining repair pathway choice. Cellular environments that disfavor resection enable Ku70–Ku80 retention at the DNA end, leading to classical non-homologous end joining (cNHEJ). A pro-resection environment favours Ku70–Ku80 displacement and the engagement of homologous recombination (HR). Error-prone pathways such as alternative end joining (aEJ) and single strand annealing (SSA) can act opportunistically on complex DNA ends or on recombination intermediates, hijacking the conservative HR process and leading to chromosome rearrangements.

Figure 4.. A decision tree of homologous recombination.

The schematic depicts the key role of the second end of the DNA double-stranded break (DSB) in determining the outcome of the homologous recombination (HR) process. Conservative outcomes are possible only if the second end is engaged for HR termination. The absence of a second end, or a failure to engage it in a timely fashion, leads to error-prone replicative HR outcomes — namely, long tract gene conversion (LTGC) and break-induced replication (BIR). Displacement of the nascent strand following LTGC places the solitary 3ʹ single-stranded DNA (ssDNA) end at risk of template switching and other spurious interactions, leading to complex breakpoints and chromosome rearrangements. The mechanisms that govern pathway selection for the displaced one-ended ssDNA end are unknown.

Figure 5.. Rad51 is an ‘early responder’ at stalled forks.

The early steps of stalled fork processing for conservative homologous recombination (HR) entail bidirectional fork stalling, nascent lagging strand resection, replisome disassembly (also termed fork collapse) and asymmetric fork reversal. RAD51 acts early in stalled fork processing to facilitate fork reversal, which remodels lagging strand ‘daughter strand gaps’ into long 3´ single-stranded DNA (ssDNA) tails formed from the displaced leading daughter strand. The combination of ssDNA structural intermediates and avid BRCA-mediated RAD51-loading activity block Ku70–Ku80 access to DNA ends at stalled forks, making HR the default repair pathway in this context. Red arrowhead indicates possible site of nuclease-mediated cleavage that could liberate a RAD51-coated DNA end for HR. Processing of the opposing fork arrested at the site of stalling generates a second DNA end and enables conservative repair by SDSA.

Figure 6.. Single strand annealing may be a conservative repair pathway at stalled replication forks.

A solitary stalled fork may undergo aberrant fork restart, with the engagement of ‘break-induced replication’ (BIR)-type copying (red). Of note, BIR in this context might not entail a DNA break intermediate. Displacement of the BIR nascent strand by the converging opposing fork results in duplication of genomic segment a, bounded, at one end, by the site of fork stalling and, at the other end, by the site at which the BIR nascent strand was displaced. A non-homologous tandem duplication forms if these two DNA ends are repaired by end joining. By contrast, repair by single strand annealing (SSA; promoted by BRCA1–BARD1) would collapse the two copies of segment a back to a single copy, thereby suppressing tandem duplication formation and maintaining normal chromosome structure.