The 3′-5′ exonucleases Hübscher, Ulrich; Shevelev, Igor V
Nature reviews. Molecular cell biology,
05/2002, Volume:
3, Issue:
5
Journal Article
Peer reviewed
Over the past few years, several new 3' 5' exonucleases have been identified. In vitro studies of these enzymes have uncovered much about their potential functions in vivo, and certain organisms with ...a defect in 3' 5' exonucleases have an increased susceptibility to cancer, especially under conditions of stress. Here, we look at not only the newly discovered enzymes, but also at the roles of other 3' 5' exonucleases in the quality control of DNA synthesis, where they act as proofreading exonucleases for DNA polymerases during DNA replication, repair and recombination.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Efficient repair of DNA double strand breaks and interstrand cross-links requires the homologous recombination (HR) pathway, a potentially error-free process that utilizes a homologous sequence as a ...repair template. A key player in HR is RAD51, the eukaryotic ortholog of bacterial RecA protein. RAD51 can polymerize on DNA to form a nucleoprotein filament that facilitates both the search for the homologous DNA sequences and the subsequent DNA strand invasion required to initiate HR. Because of its pivotal role in HR, RAD51 is subject to numerous positive and negative regulatory influences. Using a combination of molecular genetic, biochemical, and single-molecule biophysical techniques, we provide mechanistic insight into the mode of action of the FBH1 helicase as a regulator of RAD51-dependent HR in mammalian cells. We show that FBH1 binds directly to RAD51 and is able to disrupt RAD51 filaments on DNA through its ssDNA translocase function. Consistent with this, a mutant mouse embryonic stem cell line with a deletion in the FBH1 helicase domain fails to limit RAD51 chromatin association and shows hyper-recombination. Our data are consistent with FBH1 restraining RAD51 DNA binding under unperturbed growth conditions to prevent unwanted or unscheduled DNA recombination.
Background: Homologous recombination is regulated both positively and negatively in eukaryotic cells to suppress genomic instability.
Results: FBH1 can disrupt RAD51 filaments in vitro and suppresses formation of spontaneous RAD51 foci in mammalian cells. In cells defective for FBH1, hyper-recombination is observed.
Conclusion: FBH1 is a negative regulator of homologous recombination.
Significance: RAD51 activity must be carefully controlled to preserve genomic integrity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
ATP-binding cassette (ABC) transporters are a ubiquitous class of integral membrane proteins of immense clinical interest because of their strong association with human disease and pharmacology. To ...improve our understanding of these proteins, we used membrane yeast two-hybrid technology to map the protein interactome of all of the nonmitochondrial ABC transporters in the model organism Saccharomyces cerevisiae and combined this data with previously reported yeast ABC transporter interactions in the BioGRID database to generate a comprehensive, integrated 'interactome'. We show that ABC transporters physically associate with proteins involved in an unexpectedly diverse range of functions. We specifically examine the importance of the physical interactions of ABC transporters in both the regulation of one another and in the modulation of proteins involved in zinc homeostasis. The interaction network presented here will be a powerful resource for increasing our fundamental understanding of the cellular role and regulation of ABC transporters.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Bacteria and yeast possess one RecQ helicase homolog whereas humans contain five RecQ helicases, all of which are important in preserving genome stability. Three of these, BLM, WRN and RECQL4, are ...mutated in human diseases manifesting in premature aging and cancer. We are interested in determining to which extent these RecQ helicases function cooperatively. Here, we report a novel physical and functional interaction between BLM and RECQL4. Both BLM and RECQL4 interact in vivo and in vitro. We have mapped the BLM interacting site to the N-terminus of RECQL4, comprising amino acids 361-478, and the region of BLM encompassing amino acids 1-902 interacts with RECQL4. RECQL4 specifically stimulates BLM helicase activity on DNA fork substrates in vitro. The in vivo interaction between RECQL4 and BLM is enhanced during the S-phase of the cell cycle, and after treatment with ionizing radiation. The retention of RECQL4 at DNA double-strand breaks is shortened in BLM-deficient cells. Further, depletion of RECQL4 in BLM-deficient cells leads to reduced proliferative capacity and an increased frequency of sister chromatid exchanges. Together, our results suggest that BLM and RECQL4 have coordinated activities that promote genome stability.
The DNA-polymerase-X family: controllers of DNA quality? Hübscher, Ulrich; Ramadan, Kristijan; Shevelev, Igor
Nature reviews. Molecular cell biology,
200412, 2004-Dec, 2004-12-00, 20041201, Volume:
5, Issue:
12
Journal Article
Peer reviewed
Synthesis of the genetic material of the cell is achieved by a large number of DNA polymerases. Besides replicating the genome, they are involved in DNA-repair processes. Recent studies have ...indicated that certain DNA-polymerase-X-family members can synthesize unusual DNA structures, and we propose that these DNA structures might serve as 'flag wavers' for the induction of DNA-repair and/or DNA-damage-checkpoint pathways.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Type I restriction-modification enzymes are multifunctional heteromeric complexes with DNA cleavage and ATP-dependent DNA translocation activities located on motor subunit HsdR. Functional coupling ...of DNA cleavage and translocation is a hallmark of the Type I restriction systems that is consistent with their proposed role in horizontal gene transfer. DNA cleavage occurs at nonspecific sites distant from the cognate recognition sequence, apparently triggered by stalled translocation. The X-ray crystal structure of the complete HsdR subunit from E. coli plasmid R124 suggested that the triggering mechanism involves interdomain contacts mediated by ATP. In the present work, in vivo and in vitro activity assays and crystal structures of three mutants of EcoR124I HsdR designed to probe this mechanism are reported. The results indicate that interdomain engagement via ATP is indeed responsible for signal transmission between the endonuclease and helicase domains of the motor subunit. A previously identified sequence motif that is shared by the RecB nucleases and some Type I endonucleases is implicated in signaling.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
DNA polymerases (pols) catalyse the synthesis of DNA. This reaction requires a primer-template DNA in order to grow from the 3′OH end of the primer along the template. On the other hand terminal ...deoxyribonucleotidyl transferase (TdT) catalyses the addition of nucleotides at the 3′OH end of a DNA strand, without the need of a template. Pol λ and pol μ are ubiquitous enzymes, possess both DNA polymerase and terminal deoxyribonucleotidyl transferase activities and belong to pol X family, together with pol β and TdT. Here we show that pol λ, pol μ and TdT, all possess the ability to synthesise
in vitro short fragments of DNA in the absence of a primer-template or even a primer or a template in the reaction. The DNA synthesised
de novo by pol λ, pol μ and TdT appears to have an unusual structure. Furthermore we found that the amino acid Phe506 of pol λ is essential for the
de novo synthesis. This novel catalytic activity might be related to the proposed functions of these three pol X family members in DNA repair and DNA recombination.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Enzymes involved in DNA metabolic events of the highly radioresistant bacterium Deinococcus radiodurans are currently examined to understand the mechanisms that protect and repair the Deinococcus ...radiodurans genome after extremely high doses of gamma-irradiation. Although several Deinococcus radiodurans DNA repair enzymes have been characterised, no biochemical data is available for DNA ligation and DNA endhealing enzymes of Deinococcus radiodurans so far. DNA ligases are necessary to seal broken DNA backbones during replication, repair and recombination. In addition, ionizing radiation frequently leaves DNA strand-breaks that are not feasible for ligation and thus require end-healing by a 5'-polynucleotide kinase or a 3'-phosphatase. We expect that DNA ligases and end-processing enzymes play an important role in Deinococcus radiodurans DNA strand-break repair.
In this report, we describe the cloning and expression of a Deinococcus radiodurans DNA ligase in Escherichia coli. This enzyme efficiently catalyses DNA ligation in the presence of Mn(II) and NAD+ as cofactors and lysine 128 was found to be essential for its activity. We have also analysed a predicted second DNA ligase from Deinococcus radiodurans that is part of a putative DNA repair operon and shows sequence similarity to known ATP-dependent DNA ligases. We show that this enzyme possesses an adenylyltransferase activity using ATP, but is not functional as a DNA ligase by itself. Furthermore, we identified a 5'-polynucleotide kinase similar to human polynucleotide kinase that probably prepares DNA termini for subsequent ligation.
Deinococcus radiodurans contains a standard bacterial DNA ligase that uses NAD+ as a cofactor. Its enzymatic properties are similar to E. coli DNA ligase except for its preference for Mn(II) as a metal cofactor. The function of a putative second DNA ligase remains unclear, but its adenylyltransferase activity classifies it as a member of the nucleotidyltransferase family. Characterization of another protein from the same operon revealed a 5'-polynucleotide kinase with a possible role in DNA strand-break repair.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Summary
DNA polymerases of the X family have been implicated in a variety of DNA repair processes in eukaryotes. Here we show that Deinococcus radiodurans, a highly radioresistant bacterium able to ...mend hundreds of radiation‐induced double‐stranded DNA breaks, expresses a DNA polymerase belonging to the X family. This novel bacterial polymerase, named PolXDr, was identified as the product of the Deinococcal DR0467 gene. The purified PolXDr protein possesses a DNA polymerase activity that is stimulated by MnCl2, a property of the X family DNA polymerases. Antibodies raised against PolXDr recognized human pol λ, rat pol β and yeast Pol4 and, conversely, antibodies raised against these proteins recognized PolXDr. This immunological cross‐reactivity suggests a high degree of structural conservation among the polymerases of the X family. Lack of PolXDr reduced the rate of repair of double‐stranded DNA breaks and increased cell sensitivity to γ‐rays. PolXDr thus appears to play an important role in double‐stranded DNA break repair in D. radiodurans.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
RECQL4 belongs to the conserved RecQ family of DNA helicases, members of which play important roles in the maintenance of genome stability in all organisms that have been examined. Although genetic ...alterations in the RECQL4 gene are reported to be associated with three autosomal recessive disorders (Rothmund-Thomson, RAPADILINO and Baller-Gerold syndromes), the molecular role of RECQL4 still remains poorly understood. Here, we show that RECQL4 specifically interacts with the histone acetyltransferase p300 (also known as p300 HAT), both in vivo and in vitro, and that p300 acetylates one or more of the lysine residues at positions 376, 380, 382, 385 and 386 of RECQL4. Furthermore, we report that these five lysine residues lie within a short motif of 30 amino acids that is essential for the nuclear localization of RECQL4. Remarkably, the acetylation of RECQL4 by p300 in vivo leads to a significant shift of a proportion of RECQL4 protein from the nucleus to the cytoplasm. This accumulation of the acetylated RECQL4 is a result of its inability to be imported into the nucleus. Our results provide the first evidence of a post-translational modification of the RECQL4 protein, and suggest that acetylation of RECQL4 by p300 regulates the trafficking of RECQL4 between the nucleus and the cytoplasm.
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