Luis Blanco

Human DNA polymerases mu (Pol mu) and lambda (Pol lambda) are X family members involved in the repair of double-strand breaks in DNA during non-homologous end joining. Crucial abilities of these enzymes include bridging of the two 3' single-stranded overhangs and trans-polymerization using one 3' end as primer and the other as template, to minimize sequence loss. In this context, we have studied the importance of a previously uncharacterised sequence ('brooch'), located at the N-terminal boundary of the Pol ss-like polymerase core, and formed by Tyr(141), Ala(142), Cys(143), Gln(144) and Arg(145) in Pol mu, and by Trp(239), Val(240), Cys(241), Ala(242) and Gln(243) in Pol lambda. The brooch is potentially implicated in the maintenance of a closed conformation throughout the catalytic cycle, and our studies indicate that it could be a target of Cdk phosphorylation in Pol mu. The brooch is irrelevant for 1 nt gap filling, but of specific importance during end joining: single mutations in the conserved residues reduced the formation of two ended synapses and strongly diminished the ability of Pol mu and polymerase lambda to perform non-homologous end joining reactions in vitro.

DNA double-strand breaks (DSBs) are one of the most dangerous DNA lesions, since their erroneous repair by nonhomologous end-joining (NHEJ) can generate harmful chromosomal rearrangements. PolX DNA polymerases are well suited to extend DSB ends that cannot be directly ligated due to their particular ability to bind to and insert nucleotides at the imperfect template-primer structures formed during NHEJ. Herein, we have devised genetic assays in yeast to induce simultaneous DSBs in different chromosomes in vivo. The repair of these breaks in trans could result in reciprocal chromosomal translocations that were dependent on classical Ku-dependent NHEJ. End-joining events leading to translocations were mainly based on the formation of short base pairing between 3′-overhanging DNA ends coupled to gap-filling DNA synthesis. A major proportion of these events were specifically dependent on yeast DNA polymerase Pol4 activity. In addition, we have discovered that Pol4-Thr⁵⁴⁰ amino acid residue can be phosphorylated by Tel1/ATM kinase, which could modulate Pol4 activity during NHEJ. Our data suggest that the role of Tel1 in preventing break-induced chromosomal translocations can, to some extent, be due to its stimulating effect on gap-filling activity of Pol4 to repair DSBs in cis. Overall, this work provides further insight to the molecular mechanisms of DSB repair by NHEJ and presents a new perspective to the understanding of how chromosomal translocations are formed in eukaryotic cells. Chromosomal translocations are one of the most common types of genomic rearrangements, which may have a relevant impact on cell development. They are often generated from DNA double-strand breaks that are inaccurately repaired by DNA repair machinery. In this study, we have developed genetic assays in yeast to analyze the molecular mechanisms by which these translocations can arise. We found evidence showing that the classical nonhomologous end-joining repair pathway can be a source of chromosomal translocations, with a relevant role for yeast DNA polymerase Pol4 in such processes. The involvement of Pol4 is based on its efficient gap-filling DNA synthesis activity during the joining of overhanging DNA ends with short sequence complementarity. In addition, we discovered that DNA polymerase Pol4 can be modified during the repair of the breaks via phosphorylation by Tel1 kinase. This phosphorylation seems to have important structural and functional implications in the action of Pol4, which can finally influence the formation of translocations. This work provides a useful tool for deciphering factors and mechanisms involved in DNA double-strand break repair and identifying the molecular pathways leading to chromosomal translocations in eukaryotic cells.

Human DNA polymerase mu (Polμ), a family X member involved in DNA repair, has both template-directed and terminal transferase (template-independent) activities. In addition to their ability to incorporate untemplated nucleotides, another similarity between Polµ and terminal deoxynucleotidyl transferase (TdT) is their promiscuity in using ribonucleotides (NTPs), whose physiological significance is presently unknown. As shown here, Polµ can use NTPs instead of deoxynucleotides (dNTPs) during non-homologous end joining (NHEJ) of non-complementary ends, a Polµ-specific task. Moreover, a physiological concentration of Mn ²⁺ ions did benefit Polµ-mediated NHEJ by improving the efficiency and accuracy of nucleotide insertion. Analysis of different mutations in the ‘steric gate’ of the active site indicated that Polµ is taking advantage of an open active site, valid for selecting alternative activating metal ions and nucleotides as substrates. This versatility would allow ad hoc selection of the most appropriate nucleotide/metal ion combination for individual NHEJ events to gain efficiency without a cost in terms of fidelity, thus widening the spectrum of available solutions to position a discontinuous template strand in proper register for connection.

Polµ is the only DNA polymerase equipped with template-directed and terminal transferase activities. Polµ is also able to accept distortions in both primer and template strands, resulting in misinsertions and extension of realigned mismatched primer terminus. In this study, we propose a model for human Polµ-mediated dinucleotide expansion as a function of the sequence context. In this model, Polµ requires an initial dislocation, that must be subsequently stabilized, to generate large sequence expansions at different 5′-P-containing DNA substrates, including those that mimic non-homologous end-joining (NHEJ) intermediates. Our mechanistic studies point at human Polµ residues His³²⁹ and Arg³⁸⁷ as responsible for regulating nucleotide expansions occurring during DNA repair transactions, either promoting or blocking, respectively, iterative polymerization. This is reminiscent of the role of both residues in the mechanism of terminal transferase activity. The iterative synthesis performed by Polµ at various contexts may lead to frameshift mutations producing DNA damage and instability, which may end in different human disorders, including cancer or congenital abnormalities.

Non-homologous end-joining (NHEJ), the preferred pathway to repair double-strand breaks (DSBs) in higher eukaryotes, relies on a collection of molecular tools to process the broken ends, including specific DNA polymerases. Among them, Polµ is unique as it can catalyze DNA synthesis upon connection of two non-complementary ends. Here, we demonstrate that this capacity is intrinsic to Polµ, not conferred by other NHEJ factors. To understand the molecular determinants of its specific function in NHEJ, the interaction of human Polµ with DNA has been directly visualized by electromobility shift assay and footprinting assays. Stable interaction with a DNA gap requires the presence of a recessive 5′-P, thus orienting the catalytic domain for primer and nucleotide binding. Accordingly, recognition of the 5′-P is crucial to align the two DNA substrates of the NHEJ reaction. Site-directed mutagenesis demonstrates the relevance of three specific residues (Lys²⁴⁹, Arg²⁵³ and Arg⁴¹⁶) in stabilizing the primer strand during end synapsis, allowing a range of microhomology-induced distortions beneficial for NHEJ. Moreover, our results suggest that the Polµ BRCT domain, thought to be exclusively involved in interaction with NHEJ core factors, has a direct role in binding the DNA region neighbor to the 5′-P, thus boosting Polµ-mediated NHEJ reactions.

DNA polymerase lambda (Polλ) is a DNA repair polymerase, which likely plays a role in base excision repair (BER) and in non-homologous end joining (NHEJ) of DNA double-strand breaks (DSB). Here, we described a novel natural allelic variant of human Polλ (hPolλ) characterized by a single nucleotide polymorphism (SNP), C/T variation in the first base of codon 438, resulting in the amino acid change Arg to Trp. In vitro enzyme activity assays of the purified W438 Polλ variant revealed that it retained both DNA polymerization and deoxyribose phosphate (dRP) lyase activities, but had reduced base substitution fidelity. Ectopic expression of the W438 hPolλ variant in mammalian cells increases mutation frequency, affects the DSB repair NHEJ pathway, and generates chromosome aberrations. All these phenotypes are dependent upon the catalytic activity of the W438 hPolλ. The expression of a cancer-related natural variant of one specialized DNA polymerase can be associated to generic instability at the cromosomal level, probably due a defective NHEJ. These results establish that chromosomal aberrations can result from mutations in specialized DNA repair polymerases.

DNA polymerase mu (Polμ) is a family X member implicated in DNA repair, with template-directed and terminal transferase (template-independent) activities. It has been proposed that the terminal transferase activity of Polμ can be specifically required during non-homologous end joining (NHEJ) to create or increase complementarity of DNA ends. By site-directed mutagenesis in human Polμ, we have identified a specific DNA ligand residue (Arg ³⁸⁷ ) that is responsible for its limited terminal transferase activity compared to that of human TdT, its closest homologue (42% amino acid identity). Polμ mutant R387K (mimicking TdT) displayed a large increase in terminal transferase activity, but a weakened interaction with ssDNA. That paradox can be explained by the regulatory role of Arg ³⁸⁷ in the translocation of the primer from a non-productive E:DNA complex to a productive E:DNA:dNTP complex in the absence of a templating base, which is probably the rate limiting step during template-independent synthesis. Further, we show that the Polμ switch from terminal transferase to templated insertions in NHEJ reactions is triggered by recognition of a 5′-P at a second DNA end, whose 3′-protrusion could provide a templating base to facilitate such a special “pre-catalytic translocation step.” These studies shed light on the mechanism by which a rate-limited terminal transferase activity in Polμ could regulate the balance between accuracy and necessary efficiency, providing some variability during NHEJ.