José F. Ruiz

DNA polymerase mu (Pol μ), homologous to TdT, could act as a DNA mutator in eukaryotic cells

A novel DNA polymerase has been identified in human cells. Human DNA polymerase mu (Pol μ), consisting of 494 amino acids, has 41% identity to terminal deoxynucleotidyltransferase (TdT). Human Pol μ, overproduced in Escherichia coli in a soluble form and purified to homogeneity, displays intrinsic terminal deoxynucleotidyltransferase activity and a strong preference for activating Mn2+ ions. Interestingly, unlike TdT, the catalytic efficiency of polymerization carried out by Pol μ was enhanced by the presence of a template strand. Using activating Mg2+ ions, template‐enhanced polymerization was also template‐directed, leading to the preferred insertion of complementary nucleotides, although with low discrimination values. In the presence of Mn2+ ions, template‐enhanced polymerization produced a random insertion of nucleotides. Northern‐blotting and in situ analysis showed a preferential expression of Pol μ mRNA in peripheral lymphoid tissues. Moreover, a large proportion of the human expressed sequence tags corresponding to Pol μ, present in the databases, derived from germinal center B cells. Therefore, Pol μ is a good candidate to be the mutator polymerase responsible for somatic hypermutation of immunoglobulin genes.

DNA Polymerase λ, a Novel DNA Repair Enzyme in Human Cells

DNA polymerase lambda (pol λ) is a novel family X DNA polymerase that has been suggested to play a role in meiotic recombination and DNA repair. The recent demonstration of an intrinsic 5′-deoxyribose-5-phosphate lyase activity in pol λ supports a function of this enzyme in base excision repair. However, the biochemical properties of the polymerization activity of this enzyme are still largely unknown. We have cloned and purified human pol λ to homogeneity in a soluble and active form, and we present here a biochemical description of its polymerization features. In support of a role in DNA repair, pol λ inserts nucleotides in a DNA template-dependent manner and is processive in small gaps containing a 5′-phosphate group. These properties, together with its nucleotide insertion fidelity parameters and lack of proofreading activity, indicate that pol λ is a novel β-like DNA polymerase. However, the high affinity of pol λ for dNTPs (37-fold over pol β) is consistent with its possible involvement in DNA transactions occurring under low cellular levels of dNTPs. This suggests that, despite their similarities, pol β and pol λ have nonredundantin vivo functions.

A specific loop in human DNA polymerase mu allows switching between creative and DNA-instructed synthesis

Human DNA polymerase mu (Polμ) is a family X member that has terminal transferase activity but, in spite of a non-orthodox selection of the template information, displays its maximal catalytic efficiency in DNA-templated reactions. As terminal deoxynucleotidyl transferase (TdT), Polμ has a specific loop (loop1) that could provide this enzyme with its terminal transferase activity. When loop1 was deleted, human Polμ lacked TdT activity but improved DNA-binding and DNA template-dependent polymerization. Interestingly, when loop1 from TdT was inserted in Polμ (substituting its cognate loop1), the resulting chimaera displayed TdT activity, preferentially inserting dGTP residues, but had a strongly reduced template-dependent polymerization activity. Therefore, a specialized loop in Polμ, that could adopt alternative conformations, appears to provide this enzyme with a dual capacity: (i) template independency to create new DNA information, in which loop1 would have an active role by acting as a ‘pseudotemplate’; (ii) template-dependent polymerization, in which loop1 must allow binding of the template strand. Recent in vivo and in vitro data suggest that such a dual capacity could be advantageous to resolve microhomology-mediated end-joining reactions.

AID Induces Double-Strand Breaks at Immunoglobulin Switch Regions and c-MYC Causing Chromosomal Translocations in Yeast THO Mutants

Transcription of the switch (S) regions of immunoglobulin genes in B cells generates stable R-loops that are targeted by Activation Induced Cytidine Deaminase (AID), triggering class switch recombination (CSR), as well as translocations with c-MYC responsible for Burkitts lymphomas. In Saccharomyces cerevisiae, stable R-loops are formed co-transcriptionally in mutants of THO, a conserved nuclear complex involved in mRNP biogenesis. Such R-loops trigger genome instability and facilitate deamination by human AID. To understand the mechanisms that generate genome instability mediated by mRNP biogenesis impairment and by AID, we devised a yeast chromosomal system based on different segments of mammalian S regions and c-MYC for the analysis of chromosomal rearrangements in both wild-type and THO mutants. We demonstrate that AID acts in yeast at heterologous S and c-MYC transcribed sequences leading to double-strand breaks (DSBs) which in turn cause chromosomal translocations via Non-Homologous End Joining (NHEJ). AID–induced translocations were strongly enhanced in yeast THO null mutants, consistent with the idea that AID–mediated DSBs depend on R-loop formation. Our study not only provides new clues to understand the role of mRNP biogenesis in preventing genome rearrangements and the mechanism of AID-mediated genome instability, but also shows that, once uracil residues are produced by AID–mediated deamination, these are processed into DSBs and chromosomal rearrangements by the general and conserved DNA repair functions present from yeast to human cells.

Chromosomal translocations caused by either pol32-dependent or pol32-independent triparental break-induced replication.

ABSTRACT Double-strand breaks (DSBs) are harmful DNA lesions that can generate chromosomal rearrangements or chromosome losses if not properly repaired. Despite their association with a number of genetic diseases and cancer, the mechanisms by which DSBs cause rearrangements remain unknown. Using a newly developed experimental assay for the analysis of translocations occurring between two chromosomes in Saccharomyces cerevisiae, we found that a single DSB located on one chromosome uses a short homologous sequence found in a third chromosome as a bridge to complete DSB repair, leading to chromosomal translocations. Such translocations are dramatically reduced when the short homologous sequence on the third chromosome is deleted. Translocations rely on homologous recombination (HR) proteins, such as Rad51, Rad52, and Rad59, as well as on the break-induced replication-specific protein Pol32 and on Srs2, but not on Ku70. Our results indicate that a single chromosomal DSB efficiently searches for short homologous sequences throughout the genome for its repair, leading to triparental translocations between heterologous chromosomes. Given the abundance of repetitive DNA in eukaryotic genomes, the results of this study open the possibility that HR rather than nonhomologous end joining may be a major source of chromosomal translocations.

Polymerase μ is up‐regulated during the T cell‐dependent immune response and its deficiency alters developmental dynamics of spleen centroblasts

Mammalian DNA polymerase μ (Polμ), preferentially expressed in secondary lymphoid organs, is shown here to be up‐regulated in germinal centers after immunization. Alternative splicing appears to be part of Polμ regulation during an immune response. We generated Polμ‐deficient mice that are viable and show no anatomical malformation or serious alteration in lymphoid populations, with the exception of an underrepresentation of the B cell compartment. Young and aged homozygous Polμ–/– mice generated similar immune responses after immunization with the hapten (4‐hydroxy‐3‐nitrophenyl)acetyl (NP) coupled to chicken gammaglobulin (CGG), compared with their wild‐type littermates. Nonetheless, the kinetics of development of the centroblast population showed significant differences. Hypermutation analysis of the rearranged heavy chain intron region in centroblasts isolated from NP‐CGG‐immunized Polμ–/– mice showed a similar quantitative and qualitative somatic mutation spectrum, but a lower representation of heavily mutated clones. These results suggest that although it is not a critical partner, Polμ modulates the in vivo somatic hypermutation process.

Genetic and molecular analysis of mitotic recombination in Saccharomyces cerevisiae

Many systems have been developed for the study of mitotic homologous recombination (HR) in the yeast Saccharomyces cerevisiae at both genetic and molecular levels. Such systems are of great use for the analysis of different features of HR as well as of the effect of mutations, transcription, etc., on HR. Here we describe a selection of plasmid- and chromosome-borne DNA repeat assays, as well as plasmid-chromosome recombination systems, which are useful for the analysis of spontaneous and DSB-induced recombination. They can easily be used in diploid and, most importantly, in haploid yeast cells, which is a great advantage to analyze the effect of recessive mutations on HR. Such systems were designed for the analysis of a number of different HR features, which include the frequency and length of the gene conversion events, the frequency of reciprocal exchanges, the proportion of gene conversion versus reciprocal exchange, or the molecular analysis of sister chromatid exchange.

A new role for Rrm3 in repair of replication-born DNA breakage by sister chromatid recombination

Replication forks stall at different DNA obstacles such as those originated by transcription. Fork stalling can lead to DNA double-strand breaks (DSBs) that will be preferentially repaired by homologous recombination when the sister chromatid is available. The Rrm3 helicase is a replisome component that promotes replication upon fork stalling, accumulates at highly transcribed regions and prevents not only transcription-induced replication fork stalling but also transcription-associated hyper-recombination. This led us to explore the possible role of Rrm3 in the repair of DSBs when originating at the passage of the replication fork. Using a mini-HO system that induces mainly single-stranded DNA breaks, we show that rrm3Δ cells are defective in DSB repair. The defect is clearly seen in sister chromatid recombination, the major repair pathway of replication-born DSBs. Our results indicate that Rrm3 recruitment to replication-born DSBs is crucial for viability, uncovering a new role for Rrm3 in the repair of broken replication forks.

Yeast pol4 promotes tel1-regulated chromosomal translocations.

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.

Are There Mutator Polymerases

DNA polymerases are involved in different cellular events, including genome replication and DNA repair. In the last few years, a large number of novel DNA polymerases have been discovered, and the biochemical analysis of their properties has revealed a long list of intriguing features. Some of these polymerases have a very low fidelity and have been suggested to play mutator roles in different processes, like translesion synthesis or somatic hypermutation. The current view of these processes is reviewed, and the current understanding of DNA polymerases and their role as mutator enzymes is discussed.