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1.
Nat Struct Mol Biol ; 28(6): 487-500, 2021 06.
Article in English | MEDLINE | ID: mdl-34117478

ABSTRACT

Fanconi anemia (FA) is a devastating hereditary disease characterized by bone marrow failure (BMF) and acute myeloid leukemia (AML). As FA-deficient cells are hypersensitive to DNA interstrand crosslinks (ICLs), ICLs are widely assumed to be the lesions responsible for FA symptoms. Here, we show that FA-mutated cells are hypersensitive to persistent replication stress and that FA proteins play a role in the break-induced-replication (BIR)-like pathway for fork restart. Both the BIR-like pathway and ICL repair share almost identical molecular mechanisms of 53BP1-BRCA1-controlled signaling response, SLX4- and FAN1-mediated fork cleavage and POLD3-dependent DNA synthesis, suggesting that the FA pathway is intrinsically one of the BIR-like pathways. Replication stress not only triggers BMF in FA-deficient mice, but also specifically induces monosomy 7, which is associated with progression to AML in patients with FA, in FA-deficient cells.


Subject(s)
DNA Replication , Fanconi Anemia Complementation Group Proteins/physiology , Fanconi Anemia/genetics , Aneuploidy , Animals , Bone Marrow Failure Disorders/etiology , Cell Line, Transformed , Chickens , Chromosome Breakage , Chromosome Deletion , Chromosomes, Human, Pair 7/genetics , DNA Polymerase III/physiology , DNA Replication/genetics , Disease Progression , Fanconi Anemia/metabolism , Fanconi Anemia Complementation Group Proteins/deficiency , Fanconi Anemia Complementation Group Proteins/genetics , Female , HCT116 Cells , HEK293 Cells , Humans , Hydroxyurea/pharmacology , Leukemia, Myeloid, Acute/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Genetic , Species Specificity , Tumor Suppressor p53-Binding Protein 1/physiology , Ubiquitin-Protein Ligases/physiology
2.
J Mol Model ; 25(3): 80, 2019 Feb 27.
Article in English | MEDLINE | ID: mdl-30810803

ABSTRACT

Our study examines the mechanisms by which DNA polymerase (pol) δ faithfully replicates DNA. To better understand this process, we have performed all-atom molecular dynamics simulations of several DNA pol δ systems to identify conformational changes occurring prior to chemistry and investigate mechanisms by which mutations in the fingers domain (R696W and A699Q) lower fidelity. Our results indicate that, without the incoming nucleotide, a distinct open conformation occurs defined by a rotation in the fingers. The closed form, adopted when the correct nucleotide is bound, appears best organized for chemistry when three magnesium ions coordinate protein and DNA residues in the active site. Removing an unusual third metal ion from the polymerase active site causes shifting in the fingers and thumb as well as stimulating specific exonuclease ß-hairpin-DNA interactions that fray the primer terminus base pair. These changes suggest that dissociation of the third divalent ion (metal ion 'C') signals a transfer of the DNA primer from the polymerase to the exonuclease active site and implies a role for the ß-hairpin in DNA switching. Analysis of ß-hairpin movement in several systems reveals a dependence on active-site changes and suggests how Lys444 and Tyr446 present in the ß-hairpin can affect proofreading. Analysis of A699Q and R696W pol δ mutant systems reveal marked differences in the open-to-closed transition as well as ß-hairpin repositioning that explain reduced nucleotide selectivity and higher error rates.


Subject(s)
DNA Polymerase III/physiology , DNA Replication/physiology , Catalytic Domain , DNA Polymerase III/chemistry , Models, Genetic , Models, Molecular , Molecular Dynamics Simulation , Protein Domains
3.
Genet Med ; 20(8): 890-895, 2018 08.
Article in English | MEDLINE | ID: mdl-29120461

ABSTRACT

BACKGROUND: Germ-line mutations in the exonuclease domains of the POLE and POLD1 genes are associated with an increased, but yet unquantified, risk of colorectal cancer (CRC). METHODS: We identified families with POLE or POLD1 variants by searching PubMed for relevant studies prior to October 2016 and by genotyping 669 population-based CRC cases diagnosed in patients under 60 years of age, from the Australasian Colorectal Cancer Family Registry. We estimated the age-specific cumulative risks (penetrance) using a modified segregation analysis. RESULTS: We observed 67 CRCs (mean age at diagnosis = 50.2 (SD = 13.8) years) among 364 first- and second-degree relatives from 41 POLE families, and 6 CRCs (mean age at diagnosis = 39.7 (SD = 6.83) years) among 69 relatives from 9 POLD1 families. We estimated risks of CRC up to the age of 70 years (95% confidence interval) for males and females, respectively, to be 28% (95% CI, 10­42%) and 21% (95% CI, 7­33%) for POLE mutation carriers and 90% (95% CI, 33­99%) and 82% (95% CI, 26­99%) for POLD1 mutation carriers. CONCLUSION: CRC risks for POLE mutation carriers are sufficiently high to warrant consideration of colonoscopy screening and implementation of management guidelines recommended for MSH6 mutation carriers in cases of Lynch syndrome. Refinement of estimates of CRC risk for POLD1 carriers is needed; however, clinical management recommendations could follow those made for POLE carriers.


Subject(s)
Colorectal Neoplasms/genetics , DNA Polymerase III/genetics , DNA Polymerase II/genetics , Poly-ADP-Ribose Binding Proteins/genetics , Adult , Aged , DNA Polymerase II/physiology , DNA Polymerase III/physiology , Databases, Genetic , Female , Genetic Predisposition to Disease/genetics , Germ-Line Mutation/genetics , Humans , Male , Middle Aged , Pedigree , Penetrance , Poly-ADP-Ribose Binding Proteins/physiology , Risk , Risk Factors
4.
Oncogene ; 36(31): 4427-4433, 2017 08.
Article in English | MEDLINE | ID: mdl-28368425

ABSTRACT

Mutations in the POLD1 and POLE genes encoding DNA polymerases δ (Polδ) and ɛ (Polɛ) cause hereditary colorectal cancer (CRC) and have been found in many sporadic colorectal and endometrial tumors. Much attention has been focused on POLE exonuclease domain mutations, which occur frequently in hypermutated DNA mismatch repair (MMR)-proficient tumors and appear to be responsible for the bulk of genomic instability in these tumors. In contrast, somatic POLD1 mutations are seen less frequently and typically occur in MMR-deficient tumors. Their functional significance is often unclear. Here we demonstrate that expression of the cancer-associated POLD1-R689W allele is strongly mutagenic in human cells. The mutation rate increased synergistically when the POLD1-R689W expression was combined with a MMR defect, indicating that the mutator effect of POLD1-R689W results from a high rate of replication errors. Purified human Polδ-R689W has normal exonuclease activity, but the nucleotide selectivity of the enzyme is severely impaired, providing a mechanistic explanation for the increased mutation rate in the POLD1-R689W-expressing cells. The vast majority of mutations induced by the POLD1-R689W are GC→︀TA transversions and GC→︀AT transitions, with transversions showing a strong strand bias and a remarkable preference for polypurine/polypyrimidine sequences. The mutational specificity of the Polδ variant matches that of the hypermutated CRC cell line, HCT15, in which this variant was first identified. The results provide compelling evidence for the pathogenic role of the POLD1-R689W mutation in the development of the human tumor and emphasize the need to experimentally determine the significance of Polδ variants present in sporadic tumors.


Subject(s)
Colonic Neoplasms/genetics , DNA Polymerase III/genetics , Mutation , Alleles , DNA Mismatch Repair , DNA Polymerase II/genetics , DNA Polymerase III/physiology , HCT116 Cells , Humans , Hypoxanthine Phosphoribosyltransferase/genetics , Phenotype , Poly-ADP-Ribose Binding Proteins
6.
Nat Struct Mol Biol ; 22(3): 192-198, 2015 03.
Article in English | MEDLINE | ID: mdl-25664722

ABSTRACT

Three eukaryotic DNA polymerases are essential for genome replication. Polymerase (Pol) α-primase initiates each synthesis event and is rapidly replaced by processive DNA polymerases: Polɛ replicates the leading strand, whereas Polδ performs lagging-strand synthesis. However, it is not known whether this division of labor is maintained across the whole genome or how uniform it is within single replicons. Using Schizosaccharomyces pombe, we have developed a polymerase usage sequencing (Pu-seq) strategy to map polymerase usage genome wide. Pu-seq provides direct replication-origin location and efficiency data and indirect estimates of replication timing. We confirm that the division of labor is broadly maintained across an entire genome. However, our data suggest a subtle variability in the usage of the two polymerases within individual replicons. We propose that this results from occasional leading-strand initiation by Polδ followed by exchange for Polɛ.


Subject(s)
DNA Polymerase III/physiology , DNA Polymerase II/physiology , DNA Polymerase I/physiology , DNA Replication/physiology , Models, Genetic , Schizosaccharomyces/genetics , DNA/chemistry , Replication Origin
7.
Science ; 343(6166): 88-91, 2014 Jan 03.
Article in English | MEDLINE | ID: mdl-24310611

ABSTRACT

In budding yeast, one-ended DNA double-strand breaks (DSBs) and damaged replication forks are repaired by break-induced replication (BIR), a homologous recombination pathway that requires the Pol32 subunit of DNA polymerase delta. DNA replication stress is prevalent in cancer, but BIR has not been characterized in mammals. In a cyclin E overexpression model of DNA replication stress, POLD3, the human ortholog of POL32, was required for cell cycle progression and processive DNA synthesis. Segmental genomic duplications induced by cyclin E overexpression were also dependent on POLD3, as were BIR-mediated recombination events captured with a specialized DSB repair assay. We propose that BIR repairs damaged replication forks in mammals, accounting for the high frequency of genomic duplications in human cancers.


Subject(s)
DNA Breaks, Double-Stranded , DNA Polymerase III/physiology , DNA Repair/genetics , DNA Replication/genetics , Gene Duplication , Neoplasms/genetics , Cell Cycle , Cyclin E/biosynthesis , Cyclin E/genetics , DNA Polymerase III/genetics , Humans
8.
DNA Repair (Amst) ; 12(9): 691-8, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23731732

ABSTRACT

Homologous recombination (HR) is essential for maintaining genomic integrity, which is challenged by a wide variety of potentially lethal DNA lesions. Regardless of the damage type, recombination is known to proceed by RAD51-mediated D-loop formation, followed by DNA repair synthesis. Nevertheless, the participating polymerases and extension mechanism are not well characterized. Here, we present a reconstitution of this step using purified human proteins. In addition to Pol δ, TLS polymerases, including Pol η and Pol κ, also can extend D-loops. In vivo characterization reveals that Pol η and Pol κ are involved in redundant pathways for HR. In addition, the presence of PCNA on the D-loop regulates the length of the extension tracks by recruiting various polymerases and might present a regulatory point for the various recombination outcomes.


Subject(s)
DNA-Directed DNA Polymerase/chemistry , Homologous Recombination , Proliferating Cell Nuclear Antigen/chemistry , DNA Damage , DNA Polymerase III/chemistry , DNA Polymerase III/physiology , DNA Replication , DNA, Single-Stranded/biosynthesis , DNA-Directed DNA Polymerase/physiology , HeLa Cells , Humans , Osmolar Concentration , Proliferating Cell Nuclear Antigen/physiology , RNA-Binding Protein FUS/chemistry , RNA-Binding Protein FUS/physiology , Rad51 Recombinase/chemistry , DNA Polymerase iota
9.
EMBO J ; 32(9): 1334-43, 2013 May 02.
Article in English | MEDLINE | ID: mdl-23549287

ABSTRACT

DNA polymerase III (Pol III) is the catalytic α subunit of the bacterial DNA Polymerase III holoenzyme. To reach maximum activity, Pol III binds to the DNA sliding clamp ß and the exonuclease ε that provide processivity and proofreading, respectively. Here, we characterize the architecture of the Pol III-clamp-exonuclease complex by chemical crosslinking combined with mass spectrometry and biochemical methods, providing the first structural view of the trimeric complex. Our analysis reveals that the exonuclease is sandwiched between the polymerase and clamp and enhances the binding between the two proteins by providing a second, indirect, interaction between the polymerase and clamp. In addition, we show that the exonuclease binds the clamp via the canonical binding pocket and thus prevents binding of the translesion DNA polymerase IV to the clamp, providing a novel insight into the mechanism by which the replication machinery can switch between replication, proofreading, and translesion synthesis.


Subject(s)
DNA Polymerase III/metabolism , DNA Polymerase beta/metabolism , DNA Repair , DNA-Directed DNA Polymerase/chemistry , DNA/biosynthesis , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Exodeoxyribonucleases/metabolism , Multienzyme Complexes/chemistry , DNA Polymerase III/chemistry , DNA Polymerase III/genetics , DNA Polymerase III/physiology , DNA Repair/genetics , DNA Replication/genetics , DNA Replication/physiology , DNA-Directed DNA Polymerase/metabolism , DNA-Directed DNA Polymerase/physiology , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Exodeoxyribonucleases/chemistry , Exodeoxyribonucleases/genetics , Exodeoxyribonucleases/physiology , Models, Biological , Models, Molecular , Multienzyme Complexes/metabolism , Multienzyme Complexes/physiology , Protein Binding/physiology , Protein Structure, Quaternary , Protein Subunits
10.
Nat Struct Mol Biol ; 19(11): 1168-75, 2012 Nov.
Article in English | MEDLINE | ID: mdl-23064648

ABSTRACT

Although liganded nuclear receptors have been established to regulate RNA polymerase II (Pol II)-dependent transcription units, their role in regulating Pol III-transcribed DNA repeats remains largely unknown. Here we report that ~2-3% of the ~100,000-200,000 total human DR2 Alu repeats located in proximity to activated Pol II transcription units are activated by the retinoic acid receptor (RAR) in human embryonic stem cells to generate Pol III-dependent RNAs. These transcripts are processed, initially in a DICER-dependent fashion, into small RNAs (~28-65 nt) referred to as repeat-induced RNAs that cause the degradation of a subset of crucial stem-cell mRNAs, including Nanog mRNA, which modulate exit from the proliferative stem-cell state. This regulation requires AGO3-dependent accumulation of processed DR2 Alu transcripts and the subsequent recruitment of AGO3-associated decapping complexes to the target mRNA. In this way, the RAR-dependent and Pol III-dependent DR2 Alu transcriptional events in stem cells functionally complement the Pol II-dependent neuronal transcriptional program.


Subject(s)
Argonaute Proteins/metabolism , DEAD-box RNA Helicases/metabolism , Embryonic Stem Cells/physiology , RNA, Small Interfering/metabolism , Receptors, Retinoic Acid/metabolism , Ribonuclease III/metabolism , Transcription, Genetic/physiology , Alu Elements/genetics , Alu Elements/physiology , Base Sequence , Blotting, Northern , Cell Proliferation , Cells, Cultured , Chromatin Immunoprecipitation , DNA Polymerase III/physiology , Embryonic Stem Cells/metabolism , Humans , In Situ Hybridization, Fluorescence , Mass Spectrometry , Molecular Sequence Data , RNA, Messenger/genetics , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , Sequence Analysis, DNA
11.
Nucleic Acids Res ; 40(3): 1118-30, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22006845

ABSTRACT

Linear chromosomes and linear plasmids of Streptomyces are capped by terminal proteins that are covalently bound to the 5'-ends of DNA. Replication is initiated from an internal origin, which leaves single-stranded gaps at the 3'-ends. These gaps are patched by terminal protein-primed DNA synthesis. Streptomyces contain five DNA polymerases: one DNA polymerase I (Pol I), two DNA polymerases III (Pol III) and two DNA polymerases IV (Pol IV). Of these, one Pol III, DnaE1, is essential for replication, and Pol I is not required for end patching. In this study, we found the two Pol IVs (DinB1 and DinB2) to be involved in end patching. dinB1 and dinB2 could not be co-deleted from wild-type strains containing a linear chromosome, but could be co-deleted from mutant strains containing a circular chromosome. The resulting ΔdinB1 ΔdinB2 mutants supported replication of circular but not linear plasmids, and exhibited increased ultraviolet sensitivity and ultraviolet-induced mutagenesis. In contrast, the second Pol III, DnaE2, was not required for replication, end patching, or ultraviolet resistance and mutagenesis. All five polymerase genes are relatively syntenous in the Streptomyces chromosomes, including a 4-bp overlap between dnaE2 and dinB2. Phylogenetic analysis showed that the dinB1-dinB2 duplication occurred in a common actinobacterial ancestor.


Subject(s)
DNA Polymerase III/physiology , DNA Polymerase beta/physiology , DNA Replication , Streptomyces/enzymology , Streptomyces/genetics , Telomere/metabolism , Actinobacteria/genetics , Alkylation , Chromosomes, Bacterial/chemistry , Conjugation, Genetic , DNA/metabolism , DNA Damage , DNA Polymerase III/classification , DNA Polymerase III/genetics , DNA Polymerase beta/classification , DNA Polymerase beta/genetics , DNA Repair , Gene Deletion , Gene Duplication , Gene Transfer, Horizontal , Phylogeny , Plasmids/biosynthesis , Synteny , Ultraviolet Rays
12.
PLoS One ; 6(11): e27092, 2011.
Article in English | MEDLINE | ID: mdl-22073260

ABSTRACT

Mammalian DNA polymerase δ (pol δ), a four-subunit enzyme, plays a crucial and versatile role in DNA replication and various DNA repair processes. Its function as a chromosomal DNA polymerase is dependent on the association with proliferating cell nuclear antigen (PCNA) which functions as a molecular sliding clamp. All four of the pol δ subunits (p125, p50, p68, and p12) have been reported to bind to PCNA. However, the identity of the subunit of pol δ that directly interacts with PCNA and is therefore primarily responsible for the processivity of the enzyme still remains controversial. Previous model for the network of protein-protein interactions of the pol δ-PCNA complex showed that pol δ might be able to interact with a single molecule of PCNA homotrimer through its three subunits, p125, p68, and p12 in which the p50 was not included in. Here, we have confirmed that the small subunit p50 of human pol δ truthfully interacts with PCNA by the use of far-Western analysis, quantitative ELISA assay, and subcellular co-localization. P50 is required for mediation of the interaction between pol δ subassemblies and PCNA homotrimer. Thus, pol δ interacts with PCNA via its four subunits.


Subject(s)
DNA Polymerase III/physiology , Proliferating Cell Nuclear Antigen/chemistry , Base Sequence , Blotting, Western , DNA Polymerase III/chemistry , DNA Polymerase III/immunology , DNA Primers , Enzyme-Linked Immunosorbent Assay , Humans , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
13.
J Biol Chem ; 286(36): 31180-93, 2011 Sep 09.
Article in English | MEDLINE | ID: mdl-21784862

ABSTRACT

The hyperthermophilic crenarchaeon Sulfolobus solfataricus P2 encodes three B-family DNA polymerase genes, B1 (Dpo1), B2 (Dpo2), and B3 (Dpo3), and one Y-family DNA polymerase gene, Dpo4, which are related to eukaryotic counterparts. Both mRNAs and proteins of all four DNA polymerases were constitutively expressed in all growth phases. Dpo2 and Dpo3 possessed very low DNA polymerase and 3' to 5' exonuclease activities in vitro. Steady-state kinetic efficiencies (k(cat)/K(m)) for correct nucleotide insertion by Dpo2 and Dpo3 were several orders of magnitude less than Dpo1 and Dpo4. Both the accessory proteins proliferating cell nuclear antigen and the clamp loader replication factor C facilitated DNA synthesis with Dpo3, as with Dpo1 and Dpo4, but very weakly with Dpo2. DNA synthesis by Dpo2 and Dpo3 was remarkably decreased by single-stranded binding protein, in contrast to Dpo1 and Dpo4. DNA synthesis in the presence of proliferating cell nuclear antigen, replication factor C, and single-stranded binding protein was most processive with Dpo1, whereas DNA lesion bypass was most effective with Dpo4. Both Dpo2 and Dpo3, but not Dpo1, bypassed hypoxanthine and 8-oxoguanine. Dpo2 and Dpo3 bypassed uracil and cis-syn cyclobutane thymine dimer, respectively. High concentrations of Dpo2 or Dpo3 did not attenuate DNA synthesis by Dpo1 or Dpo4. We conclude that Dpo2 and Dpo3 are much less functional and more thermolabile than Dpo1 and Dpo4 in vitro but have bypass activities across hypoxanthine, 8-oxoguanine, and either uracil or cis-syn cyclobutane thymine dimer, suggesting their catalytically limited roles in translesion DNA synthesis past deaminated, oxidized base lesions and/or UV-induced damage.


Subject(s)
DNA Replication , DNA-Directed DNA Polymerase/physiology , Sulfolobus solfataricus/genetics , Bacterial Proteins/genetics , DNA/biosynthesis , DNA Damage , DNA Polymerase I/genetics , DNA Polymerase I/physiology , DNA Polymerase II/genetics , DNA Polymerase II/physiology , DNA Polymerase III/genetics , DNA Polymerase III/physiology , DNA Polymerase beta/genetics , DNA Polymerase beta/physiology , DNA-Directed DNA Polymerase/genetics
14.
Mol Cell ; 37(2): 273-81, 2010 Jan 29.
Article in English | MEDLINE | ID: mdl-20122408

ABSTRACT

We have expressed and purified 13 proteins predicted to be required for B. subtilis DNA replication. When combined with a circular DNA template with a 5' unpaired flap, these proteins reconstitute replication of both the leading and lagging strands at the physiological rate. Consistent with the in vivo requirement for two DNA polymerase III replicases for B. subtilis chromosomal replication, both PolC and DnaE are required for reconstitution of the replication fork in vitro. Leading strand synthesis requires PolC plus ten proteins; lagging strand synthesis additionally requires primase and DnaE. DnaE does not serve as the lagging strand replicase, like DNA polymerase delta in eukaryotes, but instead functions like eukaryotic DNA polymerase alpha, adding a stretch of deoxynucleotides to the RNA primer before handoff to PolC. Primase equilibrates with the fork prior to synthesis of each Okazaki fragment, and its concentration controls the frequency of initiation and Okazaki fragment size.


Subject(s)
Bacillus subtilis/genetics , Bacterial Proteins/physiology , DNA Replication/physiology , DNA-Directed DNA Polymerase/physiology , Models, Genetic , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , DNA/biosynthesis , DNA Polymerase III/genetics , DNA Polymerase III/metabolism , DNA Polymerase III/physiology , DNA-Directed DNA Polymerase/genetics , DNA-Directed DNA Polymerase/metabolism
15.
Proc Natl Acad Sci U S A ; 106(31): 12664-9, 2009 Aug 04.
Article in English | MEDLINE | ID: mdl-19617571

ABSTRACT

The actions of Escherichia coli DNA Polymerase IV (Pol IV) in mutagenesis are managed by its interaction with the beta sliding clamp. In the structure reported by Bunting et al. [EMBO J (2003) 22:5883-5892], the C-tail of Pol IV contacts a hydrophobic cleft on the clamp, while residues V303-P305 reach over the dimer interface to contact the rim of the adjacent clamp protomer. Using mutant forms of these proteins impaired for either the rim or the cleft contacts, we determined that the rim contact was dispensable for Pol IV replication in vitro, while the cleft contact was absolutely required. Using an in vitro assay to monitor Pol III*-Pol IV switching, we determined that a single cleft on the clamp was sufficient to support the switch, and that both the rim and cleft contacts were required. Results from genetic experiments support a role for the cleft and rim contacts in Pol IV function in vivo. Taken together, our findings challenge the toolbelt model and suggest instead that Pol IV contacts the rim of the clamp adjacent to the cleft that is bound by Pol III* before gaining control of the same cleft that is bound by Pol III*.


Subject(s)
DNA Polymerase III/chemistry , DNA Polymerase beta/chemistry , Escherichia coli/enzymology , 4-Nitroquinoline-1-oxide/pharmacology , DNA Polymerase III/physiology , DNA Polymerase beta/physiology , DNA Replication , Dimerization , Escherichia coli/drug effects , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Nitrofurazone/pharmacology , SOS Response, Genetics
16.
Oncogene ; 28(30): 2738-44, 2009 Jul 30.
Article in English | MEDLINE | ID: mdl-19503096

ABSTRACT

Epigenetic therapy using DNA methylation inhibitors and histone deacetylase (HDAC) inhibitors has clinical promise for the treatment of human malignancies. To investigate roles of microRNAs (miRNAs) on epigenetic therapy of gastric cancer, the miRNA expression profile was analysed in human gastric cancer cells treated with 5-aza-2'-deoxycytidine (5-Aza-CdR) and 4-phenylbutyric acid (PBA). miRNA microarray analysis shows that most of miRNAs activated by 5-Aza-CdR and PBA in gastric cancer cells are located at Alu repeats on chromosome 19. Analyses of chromatin modification show that DNA demethylation and HDAC inhibition at Alu repeats activates silenced miR-512-5p by RNA polymerase II. In addition, activation of miR-512-5p by epigenetic treatment induces suppression of Mcl-1, resulting in apoptosis of gastric cancer cells. These results suggest that chromatin remodeling at Alu repeats plays critical roles in the regulation of miRNA expression and that epigenetic activation of silenced Alu-associated miRNAs could be a novel therapeutic approach for gastric cancer.


Subject(s)
Alu Elements , Chromatin Assembly and Disassembly , Epigenesis, Genetic , MicroRNAs/physiology , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Stomach Neoplasms/therapy , Apoptosis , Azacitidine/analogs & derivatives , Azacitidine/pharmacology , Cell Line, Tumor , Chromosomes, Human, Pair 19 , DNA Methylation , DNA Polymerase II/physiology , DNA Polymerase III/physiology , Decitabine , Down-Regulation , Histone Deacetylase Inhibitors , Humans , Myeloid Cell Leukemia Sequence 1 Protein , Phenylbutyrates/pharmacology , Stomach Neoplasms/genetics , Stomach Neoplasms/pathology
17.
J Bacteriol ; 191(15): 4815-23, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19482923

ABSTRACT

Y family DNA polymerases are specialized for replication of damaged DNA and represent a major contribution to cellular resistance to DNA lesions. Although the Y family polymerase active sites have fewer contacts with their DNA substrates than replicative DNA polymerases, Y family polymerases appear to exhibit specificity for certain lesions. Thus, mutation of the steric gate residue of Escherichia coli DinB resulted in the specific loss of lesion bypass activity. We constructed variants of E. coli UmuC with mutations of the steric gate residue Y11 and of residue F10 and determined that strains harboring these variants are hypersensitive to UV light. Moreover, these UmuC variants are dominant negative with respect to sensitivity to UV light. The UV hypersensitivity and the dominant negative phenotype are partially suppressed by additional mutations in the known motifs in UmuC responsible for binding to the beta processivity clamp, suggesting that the UmuC steric gate variant exerts its effects via access to the replication fork. Strains expressing the UmuC Y11A variant also exhibit decreased UV mutagenesis. Strikingly, disruption of the dnaQ gene encoding the replicative DNA polymerase proofreading subunit suppressed the dominant negative phenotype of a UmuC steric gate variant. This could be due to a recruitment function of the proofreading subunit or involvement of the proofreading subunit in a futile cycle of base insertion/excision with the UmuC steric gate variant.


Subject(s)
DNA-Directed DNA Polymerase/physiology , Escherichia coli Proteins/physiology , Escherichia coli/radiation effects , Ultraviolet Rays , DNA Polymerase III/genetics , DNA Polymerase III/physiology , DNA-Directed DNA Polymerase/genetics , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Mutagenesis, Site-Directed , Mutation , Structure-Activity Relationship
18.
PLoS One ; 4(1): e4184, 2009.
Article in English | MEDLINE | ID: mdl-19145245

ABSTRACT

BACKGROUND: In eukaryotic cells, DNA polymerase delta (Poldelta), whose catalytic subunit p125 is encoded in the Pold1 gene, plays a central role in chromosomal DNA replication, repair, and recombination. However, the physiological role of the Poldelta in mammalian development has not been thoroughly investigated. METHODOLOGY/PRINCIPAL FINDINGS: To examine this role, we used a gene targeting strategy to generate two kinds of Pold1 mutant mice: Poldelta-null (Pold1(-/-)) mice and D400A exchanged Poldelta (Pold1(exo/exo)) mice. The D400A exchange caused deficient 3'-5' exonuclease activity in the Poldelta protein. In Poldelta-null mice, heterozygous mice developed normally despite a reduction in Pold1 protein quantity. In contrast, homozygous Pold1(-/-) mice suffered from peri-implantation lethality. Although Pold1(-/-) blastocysts appeared normal, their in vitro culture showed defects in outgrowth proliferation and DNA synthesis and frequent spontaneous apoptosis, indicating Poldelta participates in DNA replication during mouse embryogenesis. In Pold1(exo/exo) mice, although heterozygous Pold1(exo/+) mice were normal and healthy, Pold1(exo/exo) and Pold1(exo/-) mice suffered from tumorigenesis. CONCLUSIONS: These results clearly demonstrate that DNA polymerase delta is essential for mammalian early embryogenesis and that the 3'-5' exonuclease activity of DNA polymerase delta is dispensable for normal development but necessary to suppress tumorigenesis.


Subject(s)
DNA Polymerase III/physiology , Embryonic Development , Animals , DNA Replication , Embryonic Development/genetics , Exonucleases , Genotype , Mice , Mice, Mutant Strains , Neoplasms/etiology
19.
Mol Cell Biol ; 29(6): 1432-41, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19139272

ABSTRACT

Homologous recombination is an error-free mechanism for the repair of DNA double-strand breaks (DSBs). Most DSB repair events occur by gene conversion limiting loss of heterozygosity (LOH) for markers downstream of the site of repair and restricting deleterious chromosome rearrangements. DSBs with only one end available for repair undergo strand invasion into a homologous duplex DNA, followed by replication to the chromosome end (break-induced replication [BIR]), leading to LOH for all markers downstream of the site of strand invasion. Using a transformation-based assay system, we show that most of the apparent BIR events that arise in diploid Saccharomyces cerevisiae rad51Delta mutants are due to half crossovers instead of BIR. These events lead to extensive LOH because one arm of chromosome III is deleted. This outcome is also observed in pol32Delta and pol3-ct mutants, defective for components of the DNA polymerase delta (Pol delta) complex. The half crossovers formed in Pol delta complex mutants show evidence of limited homology-dependent DNA synthesis and are partially Mus81 dependent, suggesting that strand invasion occurs and the stalled intermediate is subsequently cleaved. In contrast to rad51Delta mutants, the Pol delta complex mutants are proficient for repair of a 238-bp gap by gene conversion. Thus, the BIR defect observed for rad51 mutants is due to strand invasion failure, whereas the Pol delta complex mutants are proficient for strand invasion but unable to complete extensive tracts of recombination-initiated DNA synthesis.


Subject(s)
DNA Breaks, Double-Stranded , DNA Polymerase III/physiology , DNA Repair/physiology , Rad51 Recombinase/physiology , Saccharomyces cerevisiae/physiology , Chromosome Breakage , Chromosomes, Fungal/genetics , Chromosomes, Fungal/physiology , DNA Polymerase III/genetics , DNA Repair/genetics , DNA Replication/physiology , Gene Expression Regulation, Fungal , Mutation , Rad51 Recombinase/genetics , Recombination, Genetic , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/physiology
20.
Mol Cell Biol ; 29(5): 1212-21, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19075004

ABSTRACT

DNA double-strand breaks can result from closely opposed breaks induced directly in complementary strands. Alternatively, double-strand breaks could be generated during repair of clustered damage, where the repair of closely opposed lesions has to be well coordinated. Using single and multiple mutants of Saccharomyces cerevisiae (budding yeast) that impede the interaction of DNA polymerase delta and the 5'-flap endonuclease Rad27/Fen1 with the PCNA sliding clamp, we show that the lack of coordination between these components during long-patch base excision repair of alkylation damage can result in many double-strand breaks within the chromosomes of nondividing haploid cells. This contrasts with the efficient repair of nonclustered methyl methanesulfonate-induced lesions, as measured by quantitative PCR and S1 nuclease cleavage of single-strand break sites. We conclude that closely opposed single-strand lesions are a unique threat to the genome and that repair of closely opposed strand damage requires greater spatial and temporal coordination between the participating proteins than does widely spaced damage in order to prevent the development of double-strand breaks.


Subject(s)
DNA Breaks, Double-Stranded , DNA Polymerase III/physiology , DNA Repair , Flap Endonucleases/physiology , Saccharomyces cerevisiae Proteins/physiology , Saccharomyces cerevisiae/genetics , Methyl Methanesulfonate , Mutation , Polymerase Chain Reaction
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