Ribonucleotides are frequently incorporated into DNA during eukaryotic replication. Here we map the genome-wide distribution of these ribonucleotides as markers of replication enzymology in budding yeast, using a new 5′-DNA end-mapping method, Hydrolytic End Sequencing. HydEn-Seq of DNA from ribonucleotide excision repair-deficient strains reveals replicase- and strand-specific patterns of ribonucleotides in the nuclear genome. These patterns support the role of DNA polymerases α and δ in lagging strand replication and of DNA polymerase ε in leading strand replication. They identify replication origins, termination zones and variations in ribonucleotide incorporation frequency across the genome that exceed three orders of magnitude. HydEn-Seq also reveals strand-specific 5′-DNA ends at mitochondrial replication origins, suggesting unidirectional replication of a circular genome. Given the conservation of enzymes that incorporate and process ribonucleotides in DNA, HydEn-Seq can be used to track replication enzymology in other organisms.

TREX1 is a potent 335 exonuclease that degrades singleand double-stranded DNA (ssDNA and dsDNA). TREX1 mutations at amino acid positions Asp-18 and Asp-200 in familial chilblain lupus and Aicardi-Goutières syndrome elicit dominant immune dysfunction phenotypes. Failure to appropriately disassemble genomic DNA during normal cell death processes could lead to persistent DNA signals that trigger the innate immune response and autoimmunity. We tested this concept using dsDNA plasmid and chromatin and show that the TREX1 exonuclease locates 3 termini generated by endonucleases and degrades the nicked DNA polynucleotide. A competition assay was designed using TREX1 dominant mutants and variants to demonstrate that an intact DNA binding process, coupled with dysfunctional chemistry in the active sites, explains the dominant phenotypes in TREX1 D18N, D200N, and D200H alleles. The TREX1 residues Arg-174 and Lys-175 positioned adjacent to the active sites act with the Arg-128 residues positioned in the catalytic cores to facilitate melting of dsDNA and generate ssDNA for entry into the active sites. Metal-dependent ssDNA binding in the active sites of the catalytically inactive dominant TREX1 mutants contributes to DNA retention and precludes access to DNA 3 termini by active TREX1 enzyme. Thus, the dominant disease genetics exhibited by the TREX1 D18N, D200N, and D200H alleles parallel precisely the biochemical properties of these TREX1 dimers during dsDNA degradation of plasmid and chromatin DNA in vitro. These results support the concept that failure to degrade genomic dsDNA is a principal pathway of immune activation in TREX1-mediated autoimmune disease.

Background: Mutations in the TREX1 exonuclease gene cause a spectrum of autoimmune diseases. Results: The TREX1 Arg-114 residue acts across the stable dimer interface. Conclusion: TREX1 residues in one protomer contribute to DNA degradation catalyzed in the opposing protomer. Significance: These data help to explain the heterozygous disease condition.

Background: Mutations in the TREX1 C-terminal region (CTR) cause human autoimmune disease. Results: The CTR directs TREX1 ubiquitination and interaction with ubiquilin 1. Conclusion: TREX1 ubiquitination and co-localization with ubiquilin 1 are differentially affected in autoimmune disease mutants. Significance: Multiple mechanisms of TREX1 dysfunction include altered covalent modification and diminished catalytic function, resulting in a spectrum of autoimmune diseases.

Background:The structure of TREX1 exonuclease identifies key residues positioned at the stable dimer interface. Results: The TREX1 Arg-62 acts across the dimer interface to affect DNA binding and catalysis in the opposing protomer. Conclusion: TREX1 is a functional dimer. Significance: These data help us understand how heterozygous TREX1 mutations can contribute to disease.

Ribonucleotides embedded within DNA render the DNA sensitive to the formation of single-stranded breaks under alkali conditions. Here, we describe a next-generation sequencing method called hydrolytic end sequencing (HydEn-seq) to map ribonucleotides inserted into the genome of Saccharomyce cerevisiae strains deficient in ribonucleotide excision repair. We use this method to map several genomic features in wild-type and replicase variant yeast strains.

Keratin hydrogel is shown to be hemostatic in a porcine lethal extremity hemorrhage model compared to gauze and HemCon control groups with the presumptive mechanism of action being platelet adhesion and aggregation. Results showed that keratin hydrogels reduced shock index and increased survival time in comparison to both HemCon and gauze. Experiments to elucidate the mechanism of keratin hemostasis showed that platelets readily adhered to hydrogel films and that adherent cells contained numerous pseudopods, indicative of activation. Studies employing integrin function‐blocking antibodies reduced platelet adhesion. Collectively, these studies indicate that keratin promotes hemostasis via platelet adhesion by an integrin‐mediated process. Keratin hydrogels were shown to promote hemostasis and increase survival time in a lethal hemorrhage model with platelet adhesion and activation as the likely basis for the hemostatic property of keratin biomaterials.This work was supported by the US Army, SNS Nanofiber Technologies, American Heart Association, and Mid‐Atlantic Affiliate Grant‐in‐Aid.

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