The N-terminal domain of TWINKLE contributes to single-stranded DNA binding and DNA helicase activities

The TWINKLE protein is a hexameric DNA helicase required for replication of mitochondrial DNA. TWINKLE displays striking sequence similarity to the bacteriophage T7 gene 4 protein (gp4), which is a bi-functional primase-helicase required at the phage DNA replication fork. The N-terminal domain of human TWINKLE contains some of the characteristic sequence motifs found in the N-terminal primase domain of the T7 gp4, but other important motifs are missing. TWINKLE is not an active primase in vitro and the functional role of the N-terminal region has remained elusive. In this report, we demonstrate that the N-terminal part of TWINKLE is required for efficient binding to single-stranded DNA. Truncations of this region reduce DNA helicase activity and mitochondrial DNA replisome processivity. We also find that the gp4 and TWINKLE are functionally distinct. In contrast to the phage protein, TWINKLE binds to double-stranded DNA. Moreover, TWINKLE forms stable hexamers even in the absence of Mg2+ or NTPs, which suggests that an accessory protein, a helicase loader, is needed for loading of TWINKLE onto the circular mtDNA genome

DNA precursor biosynthesis-allosteric regulation and medical applications

Ribonucleotide reductase (RNR) is a key enzyme for de novo dNTP biosynthesis. We have studied nucleotide-dependent oligomerization of the allosterically regulated mammalian RNR using a mass spectrometry–related technique called Gas-phase Electrophoretic Mobility Macromolecule Analysis (GEMMA). Our results showed that dATP and ATP induce the formation of an α6β2 protein complex. This complex can either be active or inactive depending on whether ATP or dATP is bound. In order to understand whether formation of the large complexes is a general feature in the class Ia RNRs, we compared the mammalian RNR to the E. coli enzyme. The E. coli protein is regarded a prototype for all class Ia RNRs. We found that the E. coli RNR cycles between an active α2β2 form (in the presence of ATP, dTTP or dGTP) and an inactive α4β4 form in the presence of dATP or a combination of ATP with dTTP/dGTP. The E. coli R1 mutant (H59A) which needs higher dATP concentrations to be inhibited than the wild-type enzyme had decreased ability to form these complexes. It remains to be discovered how the regulation functions in the mammalian enzyme where both the active and inactive forms are α6β2 complexes. An alternative way to produce dNTPs is via salvage biosynthesis where deoxyribonucleosides are taken up from outside the cell and phosphorylated by deoxyribonucleoside kinases. We have found that the pathogen Trypanosoma brucei, which causes African sleeping sickness, has a very efficient salvage of adenosine, deoxyadenosine and adenosine analogs such as adenine arabinoside (Ara-A). One of the conclusions made was that this nucleoside analog is phosphorylated by the T. brucei adenosine kinase and kills the parasite by causing nucleotide pool imbalances and by incorporation into nucleic acids. Ara-A-based therapies can hopefully be developed into new medicines against African sleeping sickness. Generally, the dNTPs produced from the de novo and salvage pathways can be imported into mitochondria and participate in mtDNA replication. The minimal mtDNA replisome contains DNA polymerase γA, DNA polymerase γB, helicase (TWINKLE) and the mitochondrial single-stranded DNA-binding protein (mtSSB). Here, it was demonstrated that the primase-related domain (N-terminal region) of the TWINKLE protein lacked primase activity and instead contributes to single-stranded DNA binding and DNA helicase activities. This region is not absolutely required for mitochondrial DNA replisome function but is needed for the formation of long DNA products

The N-terminal domain of TWINKLE contributes

to single-stranded DNA binding and DNA helicase activitie

ATR kinase activation in G1 phase facilitates the repair of ionizing radiation-induced DNA damage. Nucleic Acids Res

ABSTRACT The kinase ATR is activated by RPA-coated singlestranded DNA generated at aberrant replicative structures and resected double strand breaks. While many hundred candidate ATR substrates have been identified, the essential role of ATR in the replicative stress response has impeded the study of ATR kinase-dependent signalling. Using recently developed selective drugs, we show that ATR inhibition has a significantly more potent effect than ATM inhibition on ionizing radiation (IR)-mediated cell killing. Transient ATR inhibition for a short interval after IR has long-term consequences that include an accumulation of RPA foci and a total abrogation of Chk1 S345 phosphorylation. We show that ATR kinase activity in G1 phase cells is important for survival after IR and that ATR colocalizes with RPA in the absence of detectable RPA S4/8 phosphorylation. Our data reveal that, unexpectedly, ATR kinase inhibitors may be more potent cellular radiosensitizers than ATM kinase inhibitors, and that this is associated with a novel role for ATR in G1 phase cells

Cryo-EM Reveals Promoter DNA Binding and Conformational Flexibility of the General Transcription Factor TFIID

The general transcription factor IID (TFIID) is required for initiation of RNA polymerase II-dependent transcription at many eukaryotic promoters. TFIID comprises the TATA-binding protein (TBP) and several conserved TBP-associated factors (TAFs). Recognition of the core promoter by TFIID assists assembly of the preinitiation complex. Using cryo-electron microscopy in combination with methods for ab initio single-particle reconstruction and heterogeneity analysis, we have produced density maps of two conformational states of Schizosaccharomyces pombe TFIID, containing and lacking TBP. We report that TBP-binding is coupled to a massive histone-fold domain rearrangement. Moreover, docking of the TBP-TAF1(N-terminus) atomic structure to the THID map and reconstruction of a TAF-promoter DNA complex helps to account for TAF-dependent regulation of promoter-TBP and promoter-TAF interactions

S100A9-Driven Amyloid-Neuroinflammatory Cascade in Traumatic Brain Injury as a Precursor State for Alzheimer's Disease

Pro-inflammatory and amyloidogenic S100A9 protein is an important contributor to Alzheimer's disease (AD) pathology. Traumatic brain injury (TBI) is viewed as a precursor state for AD. Here we have shown that S100A9-driven amyloid-neuroinflammatory cascade was initiated in TBI and may serve as a mechanistic link between TBI and AD. By analyzing the TBI and AD human brain tissues, we demonstrated that in post-TBI tissues S100A9, produced by neurons and microglia, becomes drastically abundant compared to A beta and contributes to both precursor-plaque formation and intracellular amyloid oligomerization. Conditions implicated in TBI, such as elevated S100A9 concentration, acidification and fever, provide strong positive feedback for S100A9 nucleation-dependent amyloid formation and delay in its proteinase clearance. Consequently, both intracellular and extracellular S100A9 oligomerization correlated with TBI secondary neuronal loss. Common morphology of TBI and AD plaques indicated their similar initiation around multiple aggregation centers. Importantly, in AD and TBI we found S100A9 plaques without A beta. S100A9 and A beta plaque pathology was significantly advanced in AD cases with TBI history at earlier age, signifying TBI as a risk factor. These new findings highlight the detrimental consequences of prolonged post-TBI neuroinflammation, which can sustain S100A9-driven amyloid-neurodegenerative cascade as a specific mechanism leading to AD development