Posttranslational Regulation of Human DNA Polymerase ι

Human DNA polymerases (pols) η and ι are Y-family DNA polymerase paralogs that facilitate translesion synthesis past damaged DNA. Both polη and polι can be monoubiquitinated in vivo. Polη has been shown to be ubiquitinated at one primary site. When this site is unavailable, three nearby lysines may become ubiquitinated. In contrast, mass spectrometry analysis of monoubiquitinated polι revealed that it is ubiquitinated at over 27 unique sites. Many of these sites are localized in different functional domains of the protein, including the catalytic polymerase domain, the proliferating cell nuclear antigen-interacting region, the Rev1-interacting region, and its ubiquitin binding motifs UBM1 and UBM2. Polι monoubiquitination remains unchanged after cells are exposed to DNA-damaging agents such as UV light (generating UV photoproducts), ethyl methanesulfonate (generating alkylation damage), mitomycin C (generating interstrand cross-links), or potassium bromate (generating direct oxidative DNA damage). However, when exposed to naphthoquinones, such as menadione and plumbagin, which cause indirect oxidative damage through mitochondrial dysfunction, polι becomes transiently polyubiquitinated via Lys¹¹- and Lys⁴⁸-linked chains of ubiquitin and subsequently targeted for degradation. Polyubiquitination does not occur as a direct result of the perturbation of the redox cycle as no polyubiquitination was observed after treatment with rotenone or antimycin A, which both inhibit mitochondrial electron transport. Interestingly, polyubiquitination was observed after the inhibition of the lysine acetyltransferase KATB3/p300. We hypothesize that the formation of polyubiquitination chains attached to polι occurs via the interplay between lysine acetylation and ubiquitination of ubiquitin itself at Lys¹¹ and Lys⁴⁸ rather than oxidative damage per se.     

The response of the pediatric head to impacts onto a rigid surface

The study of pediatric head injury relies heavily on the use of finite element models and child anthropomorphic test devices (ATDs). However, these tools, in the context of pediatric head injury, have yet to be validated due to a paucity of pediatric head response data. The goal of this study is to investigate the response and injury tolerance of the pediatric head to impact.Twelve pediatric heads were impacted in a series of drop tests. The heads were dropped onto five impact locations (forehead, occiput, vertex and right and left parietal) from drop heights of 15 and 30 cm. The head could freely fall without rotation onto a flat 19 mm thick platen. The impact force was measured using a 3-axis piezoelectric load cell attached to the platen.Age and drop height were found to be significant factors in the impact response of the pediatric head. The head acceleration (14%–15 cm; 103–30 cm), Head Injury Criterion (HIC) (253%–15 cm; 154%–30 cm) and impact stiffness (5800%–15 cm; 3755%–30 cm) when averaged across all impact locations increased with age from 33 weeks gestation to 16 years, while the pulse duration (66%–15 cm; 53%–30 cm) decreased with age. Increases in head acceleration, HIC and impact stiffness were also observed with increased drop height, while pulse duration decreased with increased drop height.One important observation was that three of the four cadaveric heads between the ages of 5-months and 22-months sustained fractures from the 15 cm and 30 cm drop heights. The 5-month-old sustained a right parietal linear fracture while the 11- and 22-month-old sustained diastatic linear fractures.     

Gliotransmission and adenosine signaling promote axon regeneration

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Near-Complete Structure and Model of Tel1ATM from Chaetomium thermophilum Reveals a Robust Autoinhibited ATP State

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Immunogold Labeling of Terminal Cellulose-Synthesizing Complexes

Received 20 September 2001/ Accepted in revised form 10 October 2001     

Structure Basis for Shaping the Nse4 Protein by the Nse1 and Nse3 Dimer within the Smc5/6 Complex

The Smc5/6 complex facilitates chromosome replication and DNA break repair. Within this complex, a subcomplex composed of Nse1, Nse3 and Nse4 is thought to play multiple roles through DNA binding and regulating ATP-dependent activities of the complex. However, how the Nse1-Nse3-Nse4 subcomplex carries out these multiple functions remain unclear. To address this question, we determine the crystal structure of the Xenopus laevis Nse1-Nse3-Nse4 subcomplex at 1.7 Å resolution and examine how it interacts with DNA. Our structural analyses show that the Nse1-Nse3 dimer adopts a closed conformation and forms three interfaces with a segment of Nse4, forcing it into a Z-shaped conformation. The Nse1-Nse3-Nse4 structure provides an explanation for how the lung disease immunodeficiency and chromosome breakage syndrome-causing mutations could dislodge Nse4 from Nse1-Nse3. Our DNA binding and mutational analyses reveal that the N-terminal and the middle region of Nse4 contribute to DNA interaction and cell viability. Integrating our data with previous crosslink mass spectrometry data, we propose potential roles of the Nse1-Nse3-Nse4 complex in binding DNA within the Smc5/6 complex.     

Structural Insights into the Mechanism of Base Excision by MBD4

DNA glycosylases remove damaged or modified nucleobases by cleaving the N-glycosyl bond and the correct nucleotide is restored through subsequent base excision repair. In addition to excising threatening lesions, DNA glycosylases contribute to epigenetic regulation by mediating DNA demethylation and perform other important functions. However, the catalytic mechanism remains poorly defined for many glycosylases, including MBD4 (methyl-CpG binding domain IV), a member of the helix-hairpin-helix (HhH) superfamily. MBD4 excises thymine from G·T mispairs, suppressing mutations caused by deamination of 5-methylcytosine, and it removes uracil and modified uracils (e.g., 5-hydroxymethyluracil) mispaired with guanine. To investigate the mechanism of MBD4 we solved high-resolution structures of enzyme-DNA complexes at three stages of catalysis. Using a non-cleavable substrate analog, 2′-deoxy-pseudouridine, we determined the first structure of an enzyme-substrate complex for wild-type MBD4, which confirms interactions that mediate lesion recognition and suggests that a catalytic Asp, highly conserved in HhH enzymes, binds the putative nucleophilic water molecule and stabilizes the transition state. Observation that mutating the Asp (to Gly) reduces activity by 2700-fold indicates an important role in catalysis, but probably not one as the nucleophile in a double-displacement reaction, as previously suggested. Consistent with direct-displacement hydrolysis, a structure of the enzyme-product complex indicates a reaction leading to inversion of configuration. A structure with DNA containing 1-azadeoxyribose models a potential oxacarbenium-ion intermediate and suggests the Asp could facilitate migration of the electrophile towards the nucleophilic water. Finally, the structures provide detailed snapshots of the HhH motif, informing how these ubiquitous metal-binding elements mediate DNA binding.     

Apparent gene conversion in an Escherichia coli rec+ strain is explained by multiple rounds of reciprocal crossing-over

Summary Gene conversion, the non-reciprocal transfer of sequence information between homologous DNA sequences, has been reported in lower eukaryotes, mammals and in Escherichia coli. In an E. coli rec ⁺ strain, we established a plasmid carrying two different deleted neo genes (neoDL and neoDR) in an inverted orientation and then selected for homologous recombination events that had reconstructed an intact neo ⁺ gene. We found some plasmids that had apparently experienced intramolecular gene conversion. Further evidence, however, suggests that they are products of multiple rounds of reciprocal crossing-over,apparently involving two plasmid molecules. First, most of the Neo⁺ clones contained multiple types of Neo⁺ plasmids, although the frequency of producing the neo ⁺ clones was low. Second, all the neo ⁺ clones also contained, as a minority, one particular form of dimer, which can be formed by reciprocal crossing-over between neoDL of one plasmid molecule and neoDR of another plasmid molecule. Third, in reconstruction experiments, we cloned and purified this dimer and transferred it back into the rec ⁺ cells. The dimer gave rise to clones containing multiple types of neo ⁺ recombinant monomers, including those apparent gene conversion types, and containing only few molecules of this dimer plasmid.     

Recombination and the immortality of the germ line

Asexual populations irreversibly accumulate mildly deleterious mutations through the occasional stochastic loss of their least-loaded line, a process known as “Muller's Ratchet”. This paper explores the dynamics of this process, and the role of recombination in halting the Ratchet. Simulation studies show that an optimal class comprising n_o individuals is lost in about 10n_o generations, implying that adaptedness may deteriorate rather rapidly in geological time. Asexual organisms will persist only if they are very numerous, or if they have very small genomes, or if there is extensive negative interaction among nonallelic mutations. Otherwise, long-term persistence requires that unloaded genomes be continually generated by recombination. An approximate expression for the rate of recombination needed to halt the Ratchet is developed, and shows that substantial recombination is necessary in populations of fewer than about 10¹⁰ individuals. A further complication is introduced by mutations in sequences which specify proofreading enzymes. Since these will reduce the fidelity of their own replication, a process of positive feedback leading to an ever-accelerating loss of function is conceivable.