Is homologous recombination really an error-free process?

Abstract

Homologous recombination (HR) is an evolutionarily conserved process that plays a pivotal role in the equilibrium between genetic stability and diversity. HR is commonly considered to be error-free, but several studies have shown that HR can be error-prone. Here, we discuss the actual accuracy of HR. First, we present the product of genetic exchanges (gene conversion, GC, and crossing over, CO) and the mechanisms of HR during double strand break repair and replication restart. We discuss the intrinsic capacities of HR to generate genome rearrangements by GC or CO, either during DSB repair or replication restart. During this process, abortive HR intermediates generate genetic instability and cell toxicity. In addition to genome rearrangements, HR also primes error-prone DNA synthesis and favors mutagenesis on single stranded DNA, a key DNA intermediate during the HR process. The fact that cells have developed several mechanisms protecting against HR excess emphasize its potential risks. Consistent with this duality, several pro-oncogenic situations have been consistently associated with either decreased or increased HR levels. Nevertheless, this versatility also has advantages that we outline here. We conclude that HR is a double-edged sword, which on one hand controls the equilibrium between genome stability and diversity but, on the other hand, can jeopardize the maintenance of genomic integrity. Therefore, whether non-homologous end joining (which, in contrast with HR, is not intrinsically mutagenic) or HR is the more mutagenic process is a question that should be re-evaluated. Both processes can be "Dr. Jekyll" in maintaining genome stability/variability and "Mr. Hyde" in jeopardizing genome integrity.

Keywords: DNA double strand break repair; Homologous recombination; genetic instability; genetic variability; mutagenesis; replication stress.

Figure 1

(A) The products of HR. Gene conversion (left panel) leading to non-reciprocal exchange of a DNA sequence (in red). Crossing over (right panel): reciprocal exchanges of adjacent sequences (black and red). Note that gene conversion can be associated with or without crossing over. (B) The double-strand break repair models through HR. Left panel: Gene conversion. After resection, the single-stranded 3′-tail invades a homologous, intact double-stranded DNA, forming a D-loop (displacement loop). This process tolerates limited imperfect sequence homologies, thus creating heteroduplex intermediates bearing mismatches (blue circle). The invading 3′-end primes DNA synthesis, which then fills in the gaps. The cruciform junctions (Holliday junctions, HJ) migrate. Resolution (or dissolution) of the HJ occurs in two different orientations (black or gray triangles), resulting in gene conversion either with or without crossing over. Middle panel: Synthesis-dependent strand annealing. Initiation is similar to that of the previous model, but the invading strand de-hybridizes and re-anneals at the other end of the injured molecule; no HJ is formed. Right panel: Break-induced replication (BIR). The initiation is similar to that of the previous models, but the synthesis continues over longer distances on the chromosome arms, even reaching the end of the chromosome. Here, there is neither resolution of the HR nor crossover. (C) Single-strand annealing (SSA). When a double-strand break is generated between two homologous sequences in tandem in the same orientation (dotted arrows), an extended single-strand resection (a) reveals two complementary DNA strands that can hybridize (b). (c) Resolution of the intermediate and gap filling complete the repair, leading to the deletion of the intergenic sequences between the initial repetitions.

Figure 2

Replication-maintenance by homologous recombination. Blue and red lines indicate parental and neo-synthesized strands, respectively. (A) Replication-restart following collapse of the replication fork. (B) Repair of a broken replication fork. (C) Repair of ssDNA gaps that are left behind the moving fork after it has encountered a DNA lesion. Star: DNA damage.

Figure 3

(A) Copy of one sequence of the donor absent on the recipient molecule. One of two homologous molecules (red and black) can contain one heterologous sequence (blue). Upon gene conversion or SDSA (see Figure 1) the heterologous (blue) sequence can be copied and transferred from the donor sequence (red) to the homologous recipient sequence (black), resulting in a genetic modification of the recipient sequence. (B) Sister chromatid exchanges. Between repeat sequences (blue boxes) without misalignment (upper panel) or with misalignment resulting in unequal sister chromatid exchanges (lower panel) and amplification and loss of genetic material. (C) Impact of gene conversion. Non-reciprocal exchange of genetic information between two heteroalleles, leading to a loss of heterozygosity (upper panel) and between a pseudogene (hatched), which often contains nonsense mutations and a gene (in red), leading to the inactivation of the latter (lower panel). (D) Chromosomal rearrangements resulting from crossing-over (CO) between repeat sequences. (1) Between homologous sequences on two chromosomes or following unequal sister chromatid exchange on the same chromosome, resulting in the amplification of one molecule and the deletion of the other. (2) Intramolecular CO between two homologous sequences in a direct orientation, resulting in the excision of the intervening sequence. (3) Intramolecular CO between two homologous sequences in an inverted orientation, resulting in the inversion of the internal fragment. (4) and (5) Inter-chromosomal CO, depending upon the orientation of the homologous sequences with respect to their centromeres (blue or red circles); this process generates a translocation (4) or a dicentric and an acentric chromosome (5).

Figure 4

(A) Chromothripsis. Chromosomal shattering into pieces and abnormal re-ligation events, resulting in intra- or inter-chromosomal rearrangements. (B) A suggested model for chromothripsis occurrence, the MMBIR (microhomology mediated break induced replication). A DNA double strand end is resected to generate a 3′ overhang that will anneal with microhomologies elsewhere in the genome to initiate replication. This mechanism can lead to more complex rearrangements if it is coupled to multiple cycles of template switches. (C) Kataegis. When mutations are expected to be distributed randomly in the genome (upper cartoon), clustered mutations were found in the genomes of several cancers (lower cartoon). (D) Where kataegis occurs. These clustered mutations were at least in part correlated with the action of DNA deaminases of the APOBEC family, which deaminate cytosines on ssDNA areas found on resected DNA ends (1), stalled transcription bubbles (2), blocked replication forks (3), or HR intermediates (4).