Role of recombination and replication fork restart in repeat instability
Introduction
Expanded tracts of repetitive DNA sequences are the cause of over 30 genetic diseases and can consist of trinucleotide or larger repetitive units [1], [2], [3], [4], [5]. The expandable repeats form stable non-B-form DNA structures which impede normal cellular processes like DNA replication and repair. Expanded trinucleotide repeats (TNRs) and other structure-forming repeats break at a greater frequency than non-repetitive DNA; types of DNA breaks that occur include nicks, gaps and double-stranded breaks (DSBs). These lesions must then be repaired in the context of the repetitive DNA. Much of the time the cell will succeed in repairing DNA damage at structure-forming repeats with fidelity, i.e. with no loss or gain of genetic material, thus preserving genome integrity. However, due to both the repetitive nature of the tract as well as the structure-forming potential, mistakes that lead to repeat expansions or contractions are relatively frequent.
There are multiple pathways that repair DNA damage that occurs within TNRs and other repetitive sequences. For example, nicks and gaps can be repaired by base excision repair (BER), or by transcription-coupled repair (TCR) within transcribed regions, both of which can generate TNR expansions (for recent reviews see [2], [5] and the review by Polyzos and McMurray in this issue). Damage that results in DSBs can be repaired by various types of end-joining, by annealing of processed ends, or by recombination-based mechanisms using either a sister chromatid or homolog as the template. In addition, recombination is a primary mechanism used in restarting stalled or collapsed replication forks and in repairing gaps left behind the replication fork. This review will summarize the current knowledge about the role of mitotic recombination in generating genomic changes within repetitive DNA. We will focus on structure-forming triplet repeats, but with comparisons to results found at other biologically relevant repeats and DNA structures.
Deletion of genes required for recombination results in increased breakage of expanded TNRs, suggesting that recombination is normally required for healing these DNA breaks [6], [7]. In replicating yeast cells, homologous recombination (HR) and ligase 4-dependent end joining (EJ) both contribute to the repair of breaks at CAG repeats [6]. Genome-wide studies to identify novel genes preventing DSBs at GAA and Alu repeats identified several recombinational repair proteins as important, among them the nuclease Mre11, whose absence increased fragility of both repeats [7], [8]. Additionally, dividing cells deficient in replication proteins exhibit cell cycle arrest and gross chromosomal rearrangements at Alu repeats because recombination intermediates cannot be resolved, which results in DSBs [8]. Failure to heal breaks at expanded TNR repeats can have dire consequences for cells. Yeast cells that lack Rad52 or Ligase 4 and have expanded CAG repeat tracts undergo frequent cell cycle arrest and cell death [9].
Traditionally, DNA repair using recombination has been considered to be an error-free form of repair. However, in actuality, recombination can be highly mutagenic and a source of genomic instability [10], [11], [12], [13]. Though they are required for repair and cell health, both HR and EJ can be mutagenic when they occur within repetitive DNA, resulting in a loss (contraction) or gain (expansion) of repeat units [14]. This is largely due to the challenges of replicating or aligning DNA across a repetitive region, especially one that has formed DNA secondary structures. These DNA structures are varied and include DNA hairpins (common in CAG/CTG and CGG/GCC repeats or inverted repeats), triplexes (formed by purine-rich repeats such as GAA/TTC) and G quadruplexes (for reviews see [5], [15], [16], [17]). Though the structures are different, the common theme is that they impede DNA transactions so that replication and repair cannot proceed with fidelity within the repetitive sequence. This inaccurate repair can lead to the incorporation of errors that can range from the aberrant insertion/deletion of DNA bases, as seen in TNR repeat genetic diseases, to genomic rearrangements and loss of heterozygosity, which are commonly seen in cancers. Historically, misalignment of alleles during meiotic crossover was shown to be a mechanism for (GCN)n repeat expansions that code for polyalanine tracts [18], but discounted as a mechanism for length changes of other TNRs, such as (CAG)n repeat tracts encoding polyglutamine. However, these early studies focused on meiotic recombination and did not explore mitotic recombination as a potential mechanism for repairing DNA damage at TNRs and causing repeat instability. The following sections will delve into the various roles of recombination during DNA repair, how each contributes to genomic maintenance of repeat sequences, and the current knowledge of how recombination pathways result in repeat instability.
Section snippets
Homology-dependent recombinational repair of forks stalled by DNA structures
Addition of repeat units by definition involves DNA synthesis. Incorporation of additional bases might arise as a result of strand slippage either during replication [19] or during fork restart [3]. DNA structures formed by repetitive DNA sequences are impediments for DNA synthesis and can cause fork stalling, or gaps behind the replication fork if bypassed. GAA/TCC triplexes and GGC/CCG repeats strongly interfere with replication progression, acting as site-specific barriers [20], [21], [22].
HR-dependent instability can cause large repeat expansions
Structure-forming DNA, including expanded CAG, CGG, GAA, and ATTCT repeats as well as palindrome-forming sequences, are natural fragile sites that cause chromosomal DSBs (reviewed in [5], [15], [90], [91], [92]). Consistently, expanded GAA/TTC repeats, Alu repeats, and internal telomeric repeats stimulate mitotic crossovers in yeast [93], [94], [95] and recombination in E. coli [96], [97], [98]. In yeast, the effect of DSB repair by HR on CAG repeat stability was assessed directly by induction
Conclusions
Many repetitive DNA sequences form secondary structures that serve as constant challenges to DNA replication and repair machineries, resulting in stalled forks, nicks, gaps, and DSBs. Recombination is an important pathway to repair these lesions, and serves as a powerful guardian of the genome. However, recombinational repair at repetitive DNA tracts can be tricky as it can also be a source of mutation, including repeat expansions and contractions. This dichotomy between recombination being a
Acknowledgements
Research in CHF’s laboratory is supported by the National Institutes of Health (award P01GM105473 and R01GM122880), National Science Foundation (MCB1330743), and Tufts University. The authors have no conflicts of interest to declare.
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Current address: Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston MA 02215, USA.