DNA breaks that affect either one (single-strand break; SSB) or simultaneously both strands (double-strand break; DSB) can arise by the attack of exogenous agents, but importantly, also as a result of normal cellular metabolism and physiological processes. SSBs constitute the most common lesions occurring in DNA, and their repair, given the existence of an undamaged template strand, is normally a very efficient process. In contrast, repair of DSBs, with both strands interrupted, is more challenging, and cells rely on either direct ligation of the ends by non-homologous end joining (NHEJ) or the use of an homologous sequence as template by homologous recombination (HR). In addition, DNA break repair does not operate isolated, but integrates within other cellular functions in a complex network known as the DNA damage response (DDR). Inefficient or inaccurate DNA break repair can compromise cell survival and genome stability. This is exemplified by several human syndromes linked to mutations in the DNA- break repair and signalling machineries, which are characterized by developmental/degenerative problems (fundamentally in the nervous system), and frequently associated with increased cancer incidence. Deciphering how cells respond to and repair DNA breaks is thus a key question in our molecular understanding of cancer, as well as developmental and degenerative disease.
In general, our research interest is to understand how DNA breaks are signalled and repaired, and how, if inefficient or aberrant, these processes can impact on human health. Within this general objective, the current focus of the laboratory is the specific influence of DNA-end structure on DSB repair. Breaks arising either spontaneously or by the action of chemicals or radiation rarely harbour 5’- phosphate 3’-hydroxyl termini, and need to be unblocked by a wide variety of DNA end-processing activities. However, these functions may be compromised or overwhelmed, resulting in breaks that can only be repaired by nucleolytic trimming of DNA ends. This is not a significant drawback for repair by HR, as its mechanism involves extensive nucleolytic degradation of DSB ends, and copying of missing information from the homologous template. In contrast, the structure of DNA ends can have a critical influence on the outcome of NHEJ, since nucleolytic activity is likely to result in incompatible DNA ends, repair of which is more complex, normally requiring the use of microhomologies to bridge DNA ends and gap-filling reactions that ultimately lead to loss and/or gain of genetic information at the repair junction. Furthermore, the incompatibility of DNA ends is also likely to increase the incidence of chromosomal translocations caused by DSB misjoining, one of the driving forces of tumorigenesis. These detrimental consequences of blocked DSBs can be particularly relevant for tissues, such as the nervous system, which, given their non-proliferative nature, lack HR and rely exclusively on NHEJ for repairing DSBs. In summary, we propose that inefficient and inaccurate repair of DSBs with blocked DNA ends is particularly hazardous for cells and organisms, and therefore, a potential determinant of DDR-linked human pathologies. However, the study of how DNA-end complexity influences the DDR and repair has been traditionally impeded by the heterogeneity in the breaks induced by most agents.
Aberrant activity of DNA topoisomerase II (TOP2), which can occur spontaneously or by the action of anticancer compounds such as etoposide, is an important source for the appearance of DSBs, which are homogeneously characterized by covalent peptide blockage of 5’-ends (Figure 1). Interestingly, TDP2 is the only known enzyme in higher eukaryotes with the physiological capacity to unblock these lesions, converting them into ligatable termini (Cortés-Ledesma 2009). Consistent with our working model, TDP2 operates in a NHEJ pathway that prevents death and genome instability in response to these DNA lesions, in cells and in vivo (Gómez-Herreros 2013). Furthermore, TDP2 loss-of-function mutations in humans are associated with neurological problems that include mental retardation, seizures and progressive cerebellar ataxia (Gómez-Herreros 2014). These results highlight the relevance of blocked DSBs in general, and TOP2-induced lesions in particular, for human health.
Furthermore, we reasoned that TOP2-induced DSBs and the specificity and uniqueness of TDP2 unblocking function provided us with an ideal scenario to induce homogeneous populations of “clean” and “blocked” DSBs by inducing TOP2 damage in TDP2-proficient and deficient genetic backgrounds respectively (Figure 2), allowing us to explore the influence of DNA-end structure on the DSB-repair process and its physiological consequences. Using this strategy we have recently shown that ATM, a key factor in the DDR to DSBs and whose loss causes the human genetic syndrome Ataxia Telangiectasia (AT), is required for efficient and accurate DSB repair, preventing cell death and genome instability, but exclusively when the ends are blocked (Álvarez-Quilón 2014). Given the pivotal role of ATM in the DDR and the paradigmatic consideration of AT within DDR syndromes, these results greatly extend the reach of our working model beyond TOP2 lesions and the highly specialized action of TDP2. We are currently exploiting this system to address the differences in the DDR to “clean” and “blocked” DSBs and the physiological consequences that these differences may have. This type of comprehensive approach, covering from detailed molecular aspects to possible pathological implications, constitutes the basis of our research.
Álvarez-Quilón A, Serrano-Benítez A, Lieberman JA, Quintero C, Sánchez-Gutiérrez D, Escudero LM, Cortés-Ledesma F (2014) ATM specifically mediates repair of double-strand breaks with blocked DNA ends Nature Communications 5:3347
Gomez-Herreros F, Schuurs-Hoeijmakers JHM, McCormack M, Greally MT, Rulten S, Romero-Granados R, Counihan TJ, Chaila E, Conroy J, Ennis S, Heinzen E, Goldstein DB, Delanty N, Cortés-Ledesma F, de Brouwer APM, Cavalleri GL, El Khamisy SF, de Vries BBA, Caldecott KW (2014) TDP2 protects transcription from abortive Topoisomerase 2 activity and maintains normal neural function Nature Genetics 46:516
Gómez-Herreros F, Romero-Granados R, Zeng Z, Álvarez-Quilón A, Quintero C, Ju L, Umans L, Vermeire L, Hyulebroeck D, Caldecott KW, Cortés-Ledesma F (2013) TDP2-dependent non-homologous end-joining protects against topoisomerase II-induced DNA breaks and genome instability in cells and in vivo PLoS Genetics 9:e1003370
Cortés-Ledesma F*, El-Khamisy SF, Zuma MC, Osborn K, Caldecott KW* (2009) A novel human 5’-tyrosyl DNA phosphodiesterase that repairs topoisomerase-mediated DNA damage Nature 461:674-678 (*corresponding author)