Damage recognition by UV damage endonuclease from Schizosaccharomyces pombe

UV damage endonuclease (UVDE) from Schizosaccharomyces pombe initiates repair of UV lesions and abasic sites by nicking the DNA 5′ to the damaged site. In this paper we show that in addition UVDE incises DNA containing a single-strand nick or gap, but that the enzymatic activity on these substrates as well as on abasic sites strongly depends on the presence of a neighbouring pyrimidine residue. This indicates that, although UVDE may have been derived from an ancestral AP endonuclease its major substrate is a UV lesion and not an AP site. We propose that UVDE rotates two nucleotides into a pocket of the protein in order to bring the scissile bond close to the active site and that purine bases are excluded from this pocket. We also show that in the DNA complex residue Tyr-358 of UVDE penetrates the DNA helix causing unstacking of two residues opposite the lesion, thereby stabilizing the protein–DNA interaction, most likely by promoting bending of the DNA. In the absence of Tyr-358 the enzyme exhibits an increased catalytic activity on UV-induced lesions, but only at a lower pH of 6.5. At physiological conditions (pH 7.5) the mutant protein completely looses its catalytic activity although it can still bind to the DNA. We propose that in addition to stabilizing the bend in the DNA the hydrophobic side chain of Tyr-358 shields the active site from exposure to the solvent.     

Involvement of a carboxylated lysine in UV damage endonuclease

UV damage endonuclease is a DNA repair enzyme that can both recognize damage such as UV lesions and introduce a nick directly 5′ to them. Recently, the crystal structure of the enzyme from Thermus thermophilus was solved. In the electron density map of this structure, unexplained density near the active site was observed at the tip of Lys229. Based on this finding, it was proposed that Lys229 is post‐translationally modified. In this article, we give evidence that this modification is a carboxyl group. By combining activity assays and X‐ray crystallography on several point mutants, we show that the carboxyl group assists in metal binding required for catalysis by donating negative charge to the metal‐coordinating residue His231. Moreover, functional and structural analysis of the K229R mutant reveals that if His231 shifts away, an increased activity results on both damaged and undamaged DNA. Taken together, the results show that T. thermophilus ultraviolet damage endonuclease is carboxylated and the modified lysine is required for proper catalysis and preventing increased incision of undamaged DNA.     

Childhood Trauma and PTSD Symptoms Increase the Risk of Cognitive Impairment in a Sample of Former Indentured Child Laborers in Old Age

A growing body of evidence suggests a link between early childhood trauma, post-traumatic stress disorder (PTSD) and higher risk for dementia in old age. The aim of the present study was to investigate the association between childhood trauma exposure, PTSD and neurocognitive function in a unique cohort of former indentured Swiss child laborers in their late adulthood. To the best of our knowledge this is the first study ever conducted on former indentured child laborers and the first to investigate the relationship between childhood versus adulthood trauma and cognitive function. According to PTSD symptoms and whether they experienced childhood trauma (CT) or adulthood trauma (AT), participants (n = 96) were categorized as belonging to one of four groups: CT/PTSD+, CT/PTSD-, AT/PTSD+, AT/PTSD-. Information on cognitive function was assessed using the Structured Interview for Diagnosis of Dementia of Alzheimer Type, Multi-infarct Dementia and Dementia of other Etiology according to ICD-10 and DSM-III-R, the Mini-Mental State Examination, and a vocabulary test. Depressive symptoms were investigated as a potential mediator for neurocognitive functioning. Individuals screening positively for PTSD symptoms performed worse on all cognitive tasks compared to healthy individuals, independent of whether they reported childhood or adulthood adversity. When controlling for depressive symptoms, the relationship between PTSD symptoms and poor cognitive function became stronger. Overall, results tentatively indicate that PTSD is accompanied by cognitive deficits which appear to be independent of earlier childhood adversity. Our findings suggest that cognitive deficits in old age may be partly a consequence of PTSD or at least be aggravated by it. However, several study limitations need to considered. Consideration of cognitive deficits when treating PTSD patients and victims of lifespan trauma (even without a diagnosis of a psychiatric condition) is crucial. Furthermore, early intervention may prevent long-term deficits in memory function and development of dementia in adulthood.     

Crystal structure of the DNA repair enzyme ultraviolet damage endonuclease

The ultraviolet damage endonuclease (UVDE) performs the initial step in an alternative excision repair pathway of UV-induced DNA damage, nicking immediately adjacent to the 5' phosphate of the damaged nucleotides. Unique for a single-protein DNA repair endonuclease, it can detect different types of damage. Here we show that Thermus thermophilus UVDE shares some essential structural features with Endo IV, an enzyme from the base excision repair pathway that exclusively nicks at abasic sites. A comparison between the structures indicates how DNA is bound by UVDE, how UVDE may recognize damage, and which of its residues are involved in catalysis. Furthermore, the comparison suggests an elegant explanation of UVDE's potential to recognize different types of damage. Incision assays including point mutants of UVDE confirmed the relevance of these conclusions.     

Electrical stimulation of the growth plate: a potential approach to an epiphysiodesis

Epiphysiodesis is an operative procedure that induces bony bridges to form across a growth plate of a bone to stop longitudinal growth. This is a very common orthopedic procedure to correct disproportional long-bone growth discrepancies; however, present techniques require an operation and anesthesia. Our study was designed to develop a minimally invasive method of epiphysiodesis by using electrical stimulation with DC current. In a rabbit model, a thin titanium electrode was inserted into a single location of the distal femoral growth plate in three groups: one without current (control), one group with a constant 10 microA (low current, LC), and one group with a 50 microA (high current, HC). The current was delivered for 2 weeks. The nontreated femur served as a control for each animal. Femur lengths were measured and comparisons were made between operated (left) and nonoperated (right) femurs. Digitized histomorphometric and volumetric analyses were performed on each growth plate, and detailed assessments were made of any morphological changes. Using length measurements, the difference in femur length was significantly larger in the HC group and not in the LC or control groups, showing bone growth inhibition at the higher current. In the HC group, bony bridges and disorganized growth plates were observed. This study shows that delivery of an electrical current of 50 microA for as little as 2 weeks can markedly affect bone growth as evidenced by changes in epiphyseal plate volume and architectural organization, and the study supports the use of this minimally invasive approach as a potential method of achieving an epiphysiodesis.