Biomedical subjects

José F Ruiz

Publications and source records attributed to José F Ruiz.

5 recordsLinked to original sources

PubMed2006

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

Human DNA polymerase mu (Polmu) 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), Polmu has a specific loop (loop1) that could provide this enzyme with its terminal transferase activity. When loop1 was deleted, human Polmu lacked TdT activity but improved DNA-binding and DNA template-dependent polymerization. Interestingly, when loop1 from TdT was inserted in Polmu (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 Polmu, 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.

Amino Acid Sequence↗

PubMed2005

Characterization of SpPol4, a unique X-family DNA polymerase in Schizosaccharomyces pombe.

As predicted by the amino acid sequence, the purified protein coded by Schizosaccharomyces pombe SPAC2F7.06c is a DNA polymerase (SpPol4) whose biochemical properties resemble those of other X family (PolX) members. Thus, this new PolX is template-dependent, polymerizes in a distributive manner, lacks a detectable 3'-->5' proofreading activity and its preferred substrates are small gaps with a 5'-phosphate group. Similarly to Polmu, SpPol4 can incorporate a ribonucleotide (rNTP) into a primer DNA. However, it is not responsible for the 1-2 rNTPs proposed to be present at the mating-type locus and those necessary for mating-type switching. Unlike Polmu, SpPol4 lacks terminal deoxynucleotidyltransferase activity and realigns the primer terminus to alternative template bases only under certain sequence contexts and, therefore, it is less error-prone than Polmu. Nonetheless, the biochemical properties of this gap-filling DNA polymerase are suitable for a possible role of SpPol4 in non-homologous end-joining. Unexpectedly based on sequence analysis, SpPol4 has deoxyribose phosphate lyase activity like Polbeta and Pollambda, and unlike Polmu, suggesting also a role of this enzyme in base excision repair. Therefore, SpPol4 is a unique enzyme whose enzymatic properties are hybrid of those described for mammalian Polbeta, Pollambda and Polmu.

Amino Acid Sequence↗

PubMed2004

Overexpression of human DNA polymerase mu (Pol mu) in a Burkitt's lymphoma cell line affects the somatic hypermutation rate.

DNA polymerase mu (Pol mu) is a DNA-dependent DNA polymerase closely related to terminal deoxynucleotidyl transferase (TdT), and prone to induce template/primer misalignments and misincorporation. In addition to a proposed general role in non-homologous end joining of double-strand breaks, its mutagenic potential and preferential expression in secondary lymphoid tissues support a role in somatic hypermutation (SHM) of immunoglobulin genes. Here, we show that human Pol mu protein is expressed in the nucleus of centroblasts obtained from human tonsils, forming a characteristic foci pattern resembling that of other DNA repair proteins in response to DNA damage. Overexpression of human Pol mu in Ramos cells, in which the SHM process is constitutive, augmented the somatic mutations specifically at the variable (V) region of the immunoglobulin genes. The nature of the mutations introduced, mostly base substitutions, supports the contribution of Pol mu to mutation of G and C residues during SHM. In vitro analysis of Pol mu misincorporation on specific templates, that mimic DNA repair intermediates and correspond to mutational hotspots, indicated that many of the mutations observed in vivo can be explained by the capacity of Pol mu to induce transient template/primer misalignments.

B-Lymphocytes↗

PubMed2003

Lack of sugar discrimination by human Pol mu requires a single glycine residue.

DNA polymerase mu (Pol mu) is a novel family X DNA polymerase that has been suggested to play a role in micro-homology mediated joining and repair of double strand breaks. We show here that human Pol mu is not able to discriminate against the 2'-OH group of the sugar moiety. It inserts rNTPs with an efficiency that is <10-fold lower than that of dNTPs, in sharp contrast with the >1000-fold discrimination characteristic of most DNA-dependent DNA polymerases. The lack of sugar discrimination by Pol mu is demonstrated by its ability to add rNTPs to both DNA and RNA primer strands, and to insert both deoxy- and ribonucleotides on growing nucleic acid chains. 3D-modelling of human Pol mu based on the available Pol beta and TdT structural information allowed us to predict candidate residues involved in sugar discrimination. Thus, a single amino acid substitution in which Gly433 residue of Pol mu was mutated to the consensus tyrosine present in Pol beta, produced a strong increase in the discrimination against ribonucleotides. The unusual capacity to insert both rNTPs and dNTPs will be discussed in the context of the predicted roles of Pol mu in DNA repair.

Amino Acid Sequence↗

PubMed2003

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.

DNA-Directed DNA Polymerase↗