Repair of DNA Breaks by Break-Induced Replication

Abstract

Double-strand DNA breaks (DSBs) are the most lethal type of DNA damage, making DSB repair critical for cell survival. However, some DSB repair pathways are mutagenic and promote genome rearrangements, leading to genome destabilization. One such pathway is break-induced replication (BIR), which repairs primarily one-ended DSBs, similar to those formed by collapsed replication forks or telomere erosion. BIR is initiated by the invasion of a broken DNA end into a homologous template, synthesizes new DNA within the context of a migrating bubble, and is associated with conservative inheritance of new genetic material. This mode of synthesis is responsible for a high level of genetic instability associated with BIR. Eukaryotic BIR was initially investigated in yeast, but now it is also actively studied in mammalian systems. Additionally, a significant breakthrough has been made regarding the role of microhomology-mediated BIR in the formation of complex genomic rearrangements that underly various human pathologies.

Keywords: ALT; BIR; CRGs; DSBs; MMBIR; alternative lengthening of telomeres; break-induced replication; complex genome rearrangements; double-strand DNA breaks; microhomology-mediated break-induced replication.

Figure 1.. DSB repair formation and repair pathways.

(a) Formation of a two-ended DSB. The lightning bolt symbolizes the effect of site-specific endonucleases, gamma-rays, or other type of damage producing DSB. (b) Formation of one-ended DSB via one of three processes: (i) replication fork stalling followed by resolution; (ii) replication through a ssDNA nick; (iii) telomere erosion leading to formation of critically short telomere. (c) Pathways of two-ended DSB repair. (i) NHEJ leading to ligation of two broken ends; (ii) MMEJ initiated by end resection followed by annealing at microhomology, removal of flaps, filling of the gaps and re-ligation. (iii) SSA, initiated by 5’-to-3’ resection of broken ends followed by annealing between homologous regions, removal of 3’-flaps, filling of gaps and re-ligation. (iv) and GC, which can result either from SDSA (left) or dHJ (right). During SDSA one end of the DSB invades a homologous template and initiates copying from the donor, followed by unwinding of the newly synthesized strand from its template, and annealing to the opposite broken end, where it serves as a template for the second strand. dHJ is initiated similarly to SDSA, but D-loop is “captured” by annealing to second broken DNA, leading to formation of a Holiday Junction, which is resolved to produce crossover or non-crossover outcome. (d) One- ended breaks are repaired by BIR proceeding by strand invasion into homologous donor followed initiation of bubble-migration DNA synthesis that can proceed until the end of the chromosome.

Figure 2.. Progression of BIR and its associated instabilities.

(a) The progression of BIR. BIR begins with 5’ to 3’ resection of the one-ended DSB exposing a 3’ ssDNA end that invades at sequence homology to form a D-loop. BIR synthesis is primed by the invaded 3’-end and synthesis progresses via a migrating bubble through the end of the chromosome, resulting in conservative inheritance. (b) Genetic Instabilities associated with BIR. (i) frameshift mutations resulting from replication slippage inside the bubble; (ii) mutations and mutation clusters (red stars) resulting from DNA damage of ssDNA accumulated behind BIR bubble; (iii) translocations resulting from ectopic invasion; (iv) half-crossovers resulting from resolution of BIR intermediate.

Figure 3.. A comprehensive model of MMBIR

Molecular mechanism of MMBIR. (a) Initiation of MMBIR by fork stalling (left) or by DNA breakage leading to a DSB (right). (b) Replication fork stalling promotes template switching guided by microhomologies, which are used to prime synthesis by translesion polymerases, or other replicative polymerases. (c) Additional template switching either restarts fork progression or continues to an ectopic location to produce rearrangements. (d) DSB initiates BIR. (e) BIR switches to MMBIR by promoting template switching at microhomologies and engaging in synthesis driven by translesion or other replicative polymerases. (f) Additional template switching may restart BIR synthesis or continue to an ectopic location to produce rearrangements.

Figure 4.. Two models of ALT in yeast.

(a) Two independent pathway model. Short telomeres resulting from telomere erosion either (left) invades into other telomeres or internal telomere sequences (ITS), which leads to formation of Type I survivors, or (right) anneals to telomere ssDNA (e.g. telomere circles) followed by rolling circle replication to form Type II survivors. The proteins required for Type I or Type II formation listed on the right. (b) Unified ALT pathway model. ALT precursors are formed by Rad51-mediated invasion of eroded telomeres into other telomeres. Rad59-mediated annealing of ssDNA in precursors to (right) telomere circles or other substrates, followed by telomere extension resulting in the formation of Type II survivors. Precursors annealing to (left) ITS sequences form Type I telomeres that are spread throughout the cell by Rad51. The Type I telomeres are stabilized by (right) annealing at telomere circles or other substrates followed by extension to form a “hybrid” telomere structure.