DNA double-strand breaks (DSBs) represent one of the most dangerous forms of DNA damage. DSBs arise following exposure to ionizing radiation or radio-mimetic chemicals, but are also generated as a byproduct of normal cellular metabolism. In addition to these accidental breaks, DSBs can be introduced in a programmed manner as part of physiological processes, such as immunoglobulin class switching in mature B lymphocytes.
Efficient and accurate repair of both accidental and programmed breaks is crucial to ensure cell survival and genome stability since inefficiently or aberrantly repaired DSBs may lead to cell death, cancer-prone genome rearrangements, and inability to carry out DNA recombination processes that are crucial for the establishment of a proper immune response.
Our lab is interested in understanding the molecular mechanisms that ensure appropriate repair of DSBs, and how deregulation of these processes leads to cancer and immunodeficiencies.
DSBs occur as a by-product of DNA replication and in response to ionizing radiation but are also generated as obligate intermediates of class switch recombination (CSR) in mature B lymphocytes. CSR is a deletional recombination reaction that changes the constant region of the antibody molecule, altering its effector function. This process has the function of generating highly efficient antibodies against pathogens, and it occurs through the regulated formation and repair of DSBs. Mammalian cells employ two major options to repair DSBs (Fig. 1). In one case, DNA ends are protected from extensive processing and directly re-ligated. This pathway is known as non-homologous end-joining (NHEJ), and it is the pathway of choice in G1 phase of the cell cycle. Alternatively, the cell can profit from the availability of a homologous DNA molecule generated by DNA replication, and repair the broken DNA by copying the information in the duplicate DNA template. This process is called homologous recombination (HR), and requires extensive resection of DNA ends to occur.
NHEJ and HR cannot always be used interchangeably. Repair by end-joining is essential for class switching in mature B lymphocytes, whereas homologous recombination is crucial to faithfully repair DSBs generated during DNA replication, and therefore it ensures preservation of genome integrity in this context. DSBs become committed to a specific repair pathway shortly after their formation as a consequence of DNA end processing. A major regulatory point of DSB repair pathway choice is the initiation of 5’-3’ resection of DNA ends, which interferes with direct ligation by NHEJ, and predisposes cells to homology-dependent repair (Fig. 2). The key regulator of DNA end processing is the DNA repair factor 53BP1, whose association with DSBs in G1 promotes NHEJ by preventing DNA end resection.
The importance of DNA end protection by 53BP1 in determining DSB repair outcome is illustrated by the deleterious consequences of its deregulation. B lymphocytes lacking a functional 53BP1 protein are unable to protect DSBs from processing, and as a consequence, they cannot engage into NHEJ-mediated repair of programmed CSR breaks. This results in impaired class switching and immunodeficiency. Therefore, DNA end protection by 53BP1 is beneficial in switching B lymphocytes (Fig. 3). On the contrary, 53BP1 ability to protect DNA ends against resection represents a barrier to homologous repair during DNA replication. Under physiological conditions, this barrier is removed by the HR protein BRCA1, which displaces 53BP1 and allows for resection and appropriate repair of DSBs by HR. BRCA1 gene is frequently mutated in hereditary breast and ovarian cancers, and the inability to inactivate DNA end protection by 53BP1 in the absence of a functional BRCA1 prevents DNA end processing and physiological repair of DNA replication-associated breaks. As a consequence, DSBs are channeled into alternative NHEJ reactions leading to aberrant chromosomal rearrangements. Accumulation of unrepaired breaks and chromosomal aberrations is known as genomic instability, which is a predisposing factor to carcinogenesis. Under these conditions, DNA end protection by 53BP1 has pathological consequences.
Regulation of 53BP1 DNA end protection activity is crucial to ensure appropriate DSB repair outcome. However, the molecular mechanisms underlying 53BP1 function in the regulation of DNA end processing remain elusive.
We have previously determined that in addition to its recruitment to the chromatin surrounding the break sites, DNA damage-induced phosphorylation of 53BP1 is required for its ability to both protect DNA ends during CSR in B cells, and to promote genomic instability in BRCA1-deficient cells (Bothmer et al., Mol Cell 2011, http://www.ncbi.nlm.nih.gov/pubmed/21549309). This finding suggested that DSB-induced phosphorylation of 53BP1 was crucial for the recruitment of yet-to-be-identified DNA end protection factors.
By using a SILAC (Stable isotope labelling by amino acids in cell culture)-based proteomic approach for phospho-dependent 53BP1 interactors, we have subsequently identified several potential candidates as 53BP1 effectors in DNA end protection, including the DNA damage response proteins Rif1, PTIP, and its stable partner Pa1 as the top candidates (Fig. 4 and Di Virgilio et al., Science 2013, http://www.ncbi.nlm.nih.gov/pubmed/23306439). Our work showed that Rif1 and PTIP are indeed recruited to DSBs viainteraction with phosphorylated 53BP1, and mediate 53BP1-dependent end protection during physiological (CSR) and pathological (genomic instability) DNA repair, respectively (Fig. 3 and Di Virgilio et al., Science 2013, http://www.ncbi.nlm.nih.gov/pubmed/23306439; Callen et al., Cell 2013, http://www.ncbi.nlm.nih.gov/pubmed/23727112).
These findings indicate that the molecular events that are required for 53BP1 to promote ligation of DNA ends during CSR and the aberrant chromosomal rearrangements in BRCA1-mutant cells are, to some extent, distinct, and that multiple 53BP1 effectors are required to mediate DNA end protection in these different repair contexts.
Our lab employs a wide variety of techniques from classic biochemistry, molecular and cellular biology approaches to state-of-the-art proteomics and genomics methodologies to dissect the pathways and mechanisms that determine the balance between DNA end resection and protection, and that ensure appropriate DSB repair outcomes.
The issue of how DSBs are channelled into the appropriate repair outcome is highly relevant to our understanding of the etiopathogenesis of immunodeficiencies and cancer. The characterization of the mechanisms underlying Rif1- and 53BP1-mediated DNA end protection in the context of switching B lymphocytes will broaden our basic knowledge of this process, and provide additional candidate genes for the identification of causative mutations in patients affected by genetically-undefined CSR immune-deficiencies. Furthermore, the high proliferative capability and programmed DNA damage incurred during CSR render mature B cells particularly susceptible to aberrant DSB repair reactions leading to chromosomal translocations, which are considered primary oncogenic events in lymphomagenesis. Therefore, the identification of novel players in DSB repair in switching B cells will provide crucial insights into the mechanisms that predispose mature B lymphocytes to malignant transformation.
The impact of the our research extends beyond B lymphocyte biology and pathology. DSB repair is a general aspect of DNA metabolism, and the identification of factors that control the balance between DSB end resection and protection will elucidate the molecular links between imbalances in this delicate equilibrium and genomic instability. In one direction, these studies will shed light on the end resection-promoting factors whose unrestricted activity may cause loss of genetic information, which is a common feature in cancer genomes. On the other end, they will elucidate the molecular basis of persistent DNA end protection function and mutagenic repair in BRCA1-mutated cells, thus significantly advancing our knowledge of the molecular factors that predispose BRCA1-mutation carriers to carcinogenesis.