PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-7 (7)
 

Clipboard (0)
None

Select a Filter Below

Journals
Year of Publication
Document Types
1.  Defects in DNA degradation revealed in crystal structures of TREX1 exonuclease mutations linked to autoimmune disease 
DNA Repair  2011;11(1):65-73.
Mutations within the human TREX1 3' exonuclease are associated with Aicardi-Goutières Syndrome (AGS) and familial chilblain lupus (FCL). Both AGS and FCL are autoimmune diseases that result in increased levels of interferon alpha and circulating antibodies to DNA. TREX1 is a member of the endoplasmic reticulum (ER)-associated SET complex and participates in granzyme A-mediated cell death to degrade nicked genomic DNA. The loss of TREX1 activity may result in the accumulation of double-stranded DNA (dsDNA) degradation intermediates that trigger autoimmune activation. The X-ray crystal structures of the TREX1 wt apoprotein, the dominant D200H, D200N and D18N homodimer mutants derived from AGS and FCL patients, as well as the recessive V201D homodimer mutant have been determined. The structures of the D200H and D200N mutant proteins reveal the enzyme has lost coordination of one of the active site metals, and the catalytic histidine (H195) is trapped in a conformation pointing away from the active site. The TREX1 D18N and V201D mutants are able to bind both metals in the active site, but with inter-metal distances that are larger than optimal for catalysis. Additionally, all of the mutant structures reveal a reduced mobility in the catalytic histidine, providing further explanation for the loss of catalytic activity. The structures of the mutant TREX1 proteins provide insight into the dysfunction relating to human disease. Additionally, the TREX1 apoprotein structure together with the previously determined wild type substrate and product structures allow us to propose a distinct mechanism for the TREX1 exonuclease.
doi:10.1016/j.dnarep.2011.10.007
PMCID: PMC3253924  PMID: 22071149
TREX1; autoimmune disease; protein structure; enzyme mechanism; lupus; exonuclease
2.  BYPASS OF N2-ETHYLGUANINE BY HUMAN DNA POLYMERASE κ† 
DNA repair  2010;10(1):56-64.
The efficiency and fidelity of nucleotide incorporation and next-base extension by DNA polymerase (pol) κ past N2-ethyl-Gua were measured using steady-state and rapid kinetic analyses. DNA pol κ incorporated nucleotides and extended 3′ termini opposite N2-ethyl-Gua with measured efficiencies and fidelities similar to that opposite Gua indicating a role for DNA pol κ at the insertion and extension steps of N2-ethyl-Gua bypass. The DNA pol κ was maximally activated to similar levels by a twenty-fold lower concentration of Mn2+ compared to Mg2+. In addition, the steady state analysis indicated that high fidelity DNA pol κ-catalyzed N2-ethyl-Gua bypass is Mg2+-dependent. Strikingly, Mn2+ activation of DNA pol κ resulted in a dramatically lower efficiency of correct nucleotide incorporation opposite both N2-ethyl-Gua and Gua compared to that detected upon Mg2+ activation. This effect is largely governed by diminished correct nucleotide binding as indicated by the high Km values for dCTP insertion opposite N2-ethyl-Gua and Gua with Mn2+ activation. A rapid kinetic analysis showed diminished burst amplitudes in the presence of Mn2+ compared to Mg2+ indicating that DNA pol κ preferentially utilizes Mg2+ activation. These kinetic data support a DNA pol κ wobble base pairing mechanism for dCTP incorporation opposite N2-ethyl-Gua. Furthermore, the dramatically different polymerization efficiencies of the Y-family DNA pols κ and ι in the presence of Mn2+ suggest a metal ion-dependent regulation in coordinating the activities of these DNA pols during translesion synthesis.
doi:10.1016/j.dnarep.2010.09.007
PMCID: PMC3010520  PMID: 20952260
Y-family DNA polymerase; Translesion Synthesis; Alkylation DNA damage; Metal ion activation; Rapid kinetic analysis; DNA polymerase κ
3.  Evaluation of the TREX1 gene in a large multi-ancestral lupus cohort 
Genes and immunity  2011;12(4):270-279.
Systemic Lupus Erythematosus (SLE) is a prototypic autoimmune disorder with a complex pathogenesis in which genetic, hormonal and environmental factors play a role. Rare mutations in the TREX1 gene, the major mammalian 3′-5′ exonuclease, have been reported in sporadic SLE cases. Some of these mutations have also been identified in a rare pediatric neurologic condition featuring an inflammatory encephalopathy known as Aicardi-Goutières syndrome (AGS). We sought to investigate the frequency of these mutations in a large multi-ancestral cohort of SLE cases and controls.
Methods
Forty single-nucleotide polymorphisms (SNPs), including both common and rare variants, across the TREX1 gene were evaluated in ∼8370 patients with SLE and ∼7490 control subjects. Stringent quality control procedures were applied and principal components and admixture proportions were calculated to identify outliers for removal from analysis. Population-based case-control association analyses were performed. P values, false discovery rate q values, and odds ratios with 95% confidence intervals were calculated.
Results
The estimated frequency of TREX1 mutations in our lupus cohort was 0.5%. Five heterozygous mutations were detected at the Y305C polymorphism in European lupus cases but none were observed in European controls. Five African cases incurred heterozygous mutations at the E266G polymorphism and, again, none were observed in the African controls. A rare homozygous R114H mutation was identified in one Asian SLE patient whereas all genotypes at this mutation in previous reports for SLE were heterozygous. Analysis of common TREX1 SNPs (MAF >10%) revealed a relatively common risk haplotype in European SLE patients with neurologic manifestations, especially seizures, with a frequency of 58% in lupus cases compared to 45% in normal controls (p=0.0008, OR=1.73, 95% CI=1.25-2.39). Finally, the presence or absence of specific autoantibodies in certain populations produced significant genetic associations. For example, a strong association with anti-nRNP was observed in the European cohort at a coding synonymous variant rs56203834 (p=2.99E-13, OR=5.2, 95% CI=3.18-8.56).
Conclusion
Our data confirm and expand previous reports and provide additional support for the involvement of TREX1 in lupus pathogenesis.
doi:10.1038/gene.2010.73
PMCID: PMC3107387  PMID: 21270825
4.  RNaseH2 mutants that cause Aicardi-Goutieres syndrome are active nucleases 
Mutations in the genes encoding the RNaseH2 and TREX1 nucleases have been identified in patients with Aicardi-Goutieres syndrome (AGS). To determine if the AGS RNaseH2 mutations result in the loss of nuclease activity, the human wild-type RNaseH2 and four mutant complexes that constitute the majority of mutations identified in AGS patients have been prepared and tested for ribonuclease H activity. The heterotrimeric structures of the mutant RNaseH2 complexes are intact. Furthermore, the ribonuclease H activities of the mutant complexes are indistinguishable from the wild-type enzyme with the exception of the RNaseH2 subunit A (Gly37Ser) mutant, which exhibits some evidence of altered nuclease specificity. These data indicate that the mechanism of RNaseH2 dysfunction in AGS cannot be simply explained by loss of ribonuclease H activity and points to a more complex mechanism perhaps mediated through altered interactions with as yet identified nucleic acids or protein partners.
doi:10.1007/s00109-008-0422-3
PMCID: PMC2852111  PMID: 19034401
Aicardi-Goutieres syndrome; RNaseH2; Nuclease; Immune activation
5.  DNA binding induces active site conformational change in the human TREX2 3′-exonuclease 
Nucleic Acids Research  2009;37(7):2411-2417.
The TREX enzymes process DNA as the major 3′→5′ exonuclease activity in mammalian cells. TREX2 and TREX1 are members of the DnaQ family of exonucleases and utilize a two metal ion catalytic mechanism of hydrolysis. The structure of the dimeric TREX2 enzyme in complex with single-stranded DNA has revealed binding properties that are distinct from the TREX1 protein. The TREX2 protein undergoes a conformational change in the active site upon DNA binding including ordering of active site residues and a shift of an active site helix. Surprisingly, even when a single monomer binds DNA, both monomers in the dimer undergo the structural rearrangement. From this we have proposed a model for DNA binding and 3′ hydrolysis for the TREX2 dimer. The structure also shows how TREX proteins potentially interact with double-stranded DNA and suggest features that might be involved in strand denaturation to provide a single-stranded substrate for the active site.
doi:10.1093/nar/gkp025
PMCID: PMC2673414  PMID: 19321497
6.  WRN exonuclease activity is blocked by DNA termini harboring 3' obstructive groups 
SUMMARY
Reactive oxygen species, generated either by cellular respiration or upon exposure to environmental agents such as ionizing radiation (IR), attack DNA to form a variety of oxidized base and sugar modifications. Accumulation of oxidative DNA damage has been associated with age-related disease as well as the aging process. Single-strand breaks harboring oxidative 3' obstructive termini, e.g. 3' phosphates and 3' phosphoglycolates, must be removed prior to DNA repair synthesis or ligation. In addition, 3' tyrosyl-linked protein damage, resulting from therapeutic agents such as camptothecin (CPT), must be processed to initiate repair. Several nucleases participate in DNA repair and the excision of 3' obstructive ends. As the protein defective in the segmental progeroid Werner syndrome (WRN) possesses 3' to 5' exonuclease activity, and Werner syndrome cells are hypersensitive to IR and CPT, we examined for WRN exonuclease activity on 3' blocking lesions. Moreover, we compared side-by-side the activity of four prominent human 3' to 5' exonucleases (WRN, APE1, TREX1, and p53) on substrates containing 3' phosphates, phosphoglycolates, and tyrosyl residues. Our studies reveal that while WRN degrades 3' hydroxyl containing substrates in a nonprocessive manner, it does not excise 3' phosphate, phosphoglycolate, or tyrosyl groups. In addition, we found that APE1 was most active at excising 3' blocking termini in comparison to the disease-related exonucleases TREX1, WRN, and p53 under identical physiological reaction conditions, and that TREX1 was the most powerful 3' to 5' exonuclease on undamaged oligonucleotide substrates.
doi:10.1016/j.mad.2006.12.005
PMCID: PMC1920796  PMID: 17224176
exonuclease; WRN; Werner syndrome; 3' damage repair
7.  Structure of the Escherichia coli DNA Polymerase III ε-HOT Proofreading Complex* 
The Journal of biological chemistry  2006;281(50):38466-38471.
The ε subunit of Escherichia coli DNA polymerase III possesses 3′-exonucleolytic proofreading activity. Within the Pol III core, ε is tightly bound between the α subunit (DNA polymerase) and θ subunit. Here, we present the crystal structure of ε in complex with HOT, the bacteriophage P1-encoded homolog of θ, at 2.1 Å resolution. The ε-HOT interface is defined by two areas of contact: an interaction of the previously unstructured N terminus of HOT with an edge of the ε central β-sheet as well as interactions between HOT and the catalytically important helix α1-loop-helix α2 motif of ε. This structure provides insight into how HOT and, by implication, θ may stabilize the ε subunit, thus promoting efficient proofreading during chromosomal replication.
doi:10.1074/jbc.M606917200
PMCID: PMC1876720  PMID: 16973612

Results 1-7 (7)