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Significance Telomerase reverse transcriptase (TERT) has a conserved central cavity near the active site. Using enzymatic and mutagenesis analyses, we provide experimental evidence that an artificially looped-out telomeric repeat in the DNA primer can be transiently accommodated in the cavity of Tribolium castaneum (tcTERT). Kinetically, tcTERT requires a minimum of 4 bp between the RNA template and DNA primer for efficient DNA synthesis. An RNA duplex downstream of the RNA-template region after a flexible linker enhances the efficiency of primer extension by tcTERT. In addition to the peripheral cavities that accommodate looped-out RNA during each telomeric repeat synthesis, the central cavity that can accommodate the looped-out DNA may aid RNA-template translocation between cycles of telomeric repeat synthesis., Telomerase synthesizes telomeres at the ends of linear chromosomes by repeated reverse transcription from a short RNA template. Crystal structures of Tribolium castaneum telomerase reverse transcriptase (tcTERT) and cryoelectron microscopy (cryo-EM) structures of human and Tetrahymena telomerase have revealed conserved features in the reverse-transcriptase domain, including a cavity near the DNA 3′ end and snug interactions with the RNA template. For the RNA template to translocate, it needs to be unpaired and separated from the DNA product. Here we investigate the potential of the structural cavity to accommodate a looped-out DNA bulge and enable the separation of the RNA/DNA hybrid. Using tcTERT as a model system, we show that a looped-out telomeric repeat in the DNA primer can be accommodated and extended by tcTERT but not by retroviral reverse transcriptase. Mutations that reduce the cavity size reduce the ability of tcTERT to extend the looped-out DNA substrate. In agreement with cryo-EM structures of telomerases, we find that tcTERT requires a minimum of 4 bp between the RNA template and DNA primer for efficient DNA synthesis. We also have determined the ternary-complex structure of tcTERT including a downstream RNA/DNA hybrid at 2.0-Å resolution and shown that a downstream RNA duplex, equivalent to the 5′ template-boundary element in telomerase RNA, enhances the efficiency of telomere synthesis by tcTERT. Although TERT has a preformed active site without the open-and-closed conformational changes, it contains cavities to accommodate looped-out RNA and DNA. The flexible RNA–DNA binding likely underlies the processivity of telomeric repeat addition.

Oxidatively induced DNA lesions 8,5′-cyclopurine-2′-deoxynucleosides (cdPus) are prevalent and cytotoxic by impeding DNA replication and transcription. Both the 5′ R - and 5′ S -diastereomers of cdPu can be removed by nucleotide excision repair; however, the 5′ S -cdPu is more resistant to repair than the 5′ R counterpart. Here, we report the crystal structures of human polymerase (Pol) η bypassing 5′ S -8,5′-cyclo-2′-deoxyadenosine (cdA) in insertion and the following two extension steps. The cdA-containing DNA structures vary in response to the protein environment. Supported by the “molecular splint” of Pol η, the structure of 5′ S -cdA at 1.75-Å resolution reveals that the backbone is pinched toward the minor groove and the adenine base is tilted. In the templating position, the cdA takes up the extra space usually reserved for the thymine dimer, and dTTP is efficiently incorporated by Pol η in the presence of Mn 2+ . Rigid distortions of the DNA duplex by cdA, however, prevent normal base pairing and hinder immediate primer extension by Pol η. Our results provide structural insights into the strong replication blockage effect and the mutagenic property of the cdPu lesions in cells.

Enzyme catalysis has been studied for over a century. How it actually occurs has not been visualized until recently. By combining in crystallo reaction and X-ray diffraction analysis of reaction intermediates, we have obtained unprecedented atomic details of the DNA synthesis process. Contrary to the established theory that enzyme-substrate complexes and transition states have identical atomic composition and catalysis occurs by the two-metal-ion mechanism, we have discovered that an additional divalent cation has to be captured en route to product formation. Unlike the canonical two metal ions, which are coordinated by DNA polymerases, this third metal ion is free of enzyme coordination. Its location between the α- and β-phosphates of dNTP suggests that the third metal ion may drive the phosphoryltransfer from the leaving group opposite to the 3′-OH nucleophile. Experimental data indicate that binding of the third metal ion may be the rate-limiting step in DNA synthesis and the free energy associated with the metal-ion binding can overcome the activation barrier to the DNA synthesis reaction. Electronic supplementary material The online version of this article (doi:10.1186/s13578-016-0118-2) contains supplementary material, which is available to authorized users.

Fruits containing high antioxidant capacities and other bioactivities are ideal for promoting longevity and health span. However, few fruits are known to improve the survival and health span in animals, let alone the underlying mechanisms. Here we investigate the effects of nectarine, a globally consumed fruit, on life span and health span in Drosophila melanogaster. Wild-type flies were fed standard, dietary restriction (DR), or high-fat diet supplemented with 0-4% nectarine extract. We measured life span, food intake, locomotor activity, fecundity, gene expression changes, and oxidative damage indicated by the level of 4-hydroxynonenal-protein adduct in these flies. We also measured life span, locomotor activity, and oxidative damage in sod1 mutant flies on the standard diet supplemented with 0-4% nectarine. Supplementation with 4% nectarine extended life span, increased fecundity, and decreased expression of some metabolic genes, including a key gluconeogenesis gene, PEPCK, and oxidative stress-response genes, including peroxiredoxins, in female wild-type flies fed the standard, DR, or high-fat diet. Nectarine reduced oxidative damage in wild-type females fed the high-fat diet. Moreover, nectarine improved the survival of and reduced oxidative damage in female sod1 mutant flies. Together, these findings suggest that nectarine promotes longevity and health span partly by modulating glucose metabolism and reducing oxidative damage.