Cyclobutane pyrimidine dimers and (6-4) photoproducts block polymerization by DNA polymerase I

Bipyrimidine cyclobutane dimers and 6-4'-(pyrimidin-2'-one)-pyrimidine photoproducts are the major adducts formed in DNA following exposure to ultraviolet light. The relationship between the type and frequency of UV-induced DNA damage and the effects of such damage on DNA replication were investigated. UV-irradiated M13 phage DNA was employed in polymerization reactions with the Kenow fragment of Escherichia coli DNA polymerase I. The locations and frequencies of polymerase termination events occurring within a defined sequence of M13 DNA were compared with measurements of the locations and frequencies of UV-induced DNA damage of the same DNA sequence by using UV-specific enzymatic and chemical methods. The results indicate that both cyclobutane dimers and (6-4) photoproducts quantitatively block polymerization by DNA polymerase I.     

The X-ray structure of a tetrapeptide bound to the active site of human cyclophilin A

Human cyclophilin A (165 residues) has peptidyl-prolyl cis-trans isomerase activity. Here we report a high-resolution three-dimensional X-ray structure of a substrate, ac-Ala-Ala-Pro-Ala-amc (ac. acetyl: amc. amidomethylcoumarin) bound to the active-site of cyclophilin. The structure consisting of a dimer of complexes and 135 water molecules was refined to a crystallographic R-factor of 17.7% for all data in the range 8 Å-2.3 Å.     

EARLY STARVATION1 specifically affects the phosphorylation action of starch‐related dikinases

Starch phosphorylation by starch‐related dikinases glucan, water dikinase (GWD) and phosphoglucan, water dikinase (PWD) is a key step in starch degradation. Little information is known about the precise structure of the glucan substrate utilized by the dikinases and about the mechanisms by which these structures may be influenced. A 50‐kDa starch‐binding protein named EARLY STARVATION1 (ESV1) was analyzed regarding its impact on starch phosphorylation. In various in vitro assays, the influences of the recombinant protein ESV1 on the actions of GWD and PWD on the surfaces of native starch granules were analyzed. In addition, we included starches from various sources as well as truncated forms of GWD. ESV1 preferentially binds to highly ordered, α‐glucans, such as starch and crystalline maltodextrins. Furthermore, ESV1 specifically influences the action of GWD and PWD at the starch granule surface. Starch phosphorylation by GWD is decreased in the presence of ESV1, whereas the action of PWD increases in the presence of ESV1. The unique alterations observed in starch phosphorylation by the two dikinases are discussed in regard to altered glucan structures at the starch granule surface.     

Brain Shuttle Antibody for Alzheimer’s Disease with Attenuated Peripheral Effector Function due to an Inverted Binding Mode

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Rescue of a tk-plasmid from transgenic mice reveals its episomal transmission

Summary This communication demonstrates the usefulness of the plamid rescue procedure for recovery of plasmids from transgenic mice. We have microinjected the plasmid pSK1 harbouring the Herpes simplex virus thymidine kinase gene into fertilized mouse oocytes and succeeded in recovering plasmids from newborns by transformation of E. coli either with HindIII cut cellular DNA or with uncut DNA. The majority of the rescued plasmids were indistinguishable from pSK1 by restriction analysis. The rescued plasmids proved to be functionally active in a transient expression assay in mouse Ltk^- cells. The pSK1 DNA sequences were inherited by up to 90% of the second generation progeny mice, which is not in agreement with a Mendelian transmission of heterozygous markers integrated into a single site of the chromosome.These data support the assumption that germ line transmission of non-integrated episomal plasmids can occur.     

Mechanism of action of a mammalian DNA repair endonuclease

The mechanism of action of a DNA repair endonuclease isolated from calf thymus was determined. The calf thymus endonuclease possesses a substrate specificity nearly identical with that of Escherichia coli endonuclease III following DNA damage by high doses of UV light, osmium tetroxide, and other oxidizing agents. The calf thymus enzyme incises damaged DNA at sites of pyrimidines. A cytosine photoproduct was found to be the primary monobasic UV adduct. The calf thymus endonuclease and E. coli endonuclease III were found to possess similar, but not identical, DNA incision mechanisms. The mechanism of action of the calf thymus endonuclease was deduced by analysis of the 3' and 5' termini of the enzyme-generated DNA scission products with DNA sequencing methodologies and HPLC analysis of the material released by the enzyme following DNA damage. The calf thymus endonuclease removes UV light and osmium tetroxide damaged bases via an N-glycosylase activity followed by a 3' apurinic/apyrimidinic (AP) endonuclease activity. The calf thymus endonuclease also possesses a novel 5' AP endonuclease activity not possessed by endonuclease III. The product of this three-step mechanism is a nucleoside-free site flanked by 3'-and 5'-terminal phosphate groups. These results indicate the conservation of both substrate specificity and mechanism of action in the enzymatic removal of oxidative base damage between prokaryotes and eukaryotes. We propose the name redoxy endonucleases for this group of enzymes.     

Specificity of the heme requirement for growth of Bacteroides ruminicola

Caldwell, D. R. (U.S. Department of Agriculture, Beltsville, Md.), D. C. White, M. P. Bryant, and R. N. Doetsch. Specificity of the heme requirement for growth of Bacteroides ruminicola. J. Bacteriol. 90:1645-1654. 1965.-Previous studies suggested that most strains of Bacteroides ruminicola subsp. ruminicola require heme for growth. Present studies with heme-requiring strain 23 showed that protoheme was replaced by various porphyrins, uroporphyrinogen, coproporphyrinogen, certain iron-free metalloporphyrins, hemes, and certain heme-proteins containing readily removable hemes. Strain 23 utilized a wider range of tetrapyrroles than hemin-requiring bacteria previously studied. Inactive compounds included porphyrin biosynthesis intermediates preceding the tetrapyrrole stage and related compounds; uroporphyrin, chlorophyll, pheophytin, phycoerythrin, bilirubin, pyrrole, FeSO(4) with or without chelating agents; and representative ferrichrome compounds. Strain 23, two other strains representing predominant biotypes of B. ruminicola subsp. ruminicola, and one closely related strain grew in media containing heme-free protoporphyrin, mesoporphyrin, hematoporphyrin, or deuteroporphyrin, apparently inserting iron into several nonvinyl porphyrins. Porphobilinogen and porphyrin synthesis, apparently via the commonly known heme synthesis pathway, occurred during growth of heme-independent B. ruminicola subsp. brevis strain GA33 in a tetrapyrrole-free medium containing delta-aminolevulinic acid, but delta-aminolevulinic acid metabolism to porphobilinogen or porphyrins could not be detected in cells of heme-requiring strain 23 grown in the same medium with hemin added. Growth of strain 23 with uroporphyrinogen, coproporphyrinogen, or protoporphyrin IX replacing hemin suggests that part of the commonly known heme-biosynthesis pathway is present in this strain, but nutritional and metabolic evidence indicates that some or all of the enzymes synthesizing the tetrapyrrole nucleus from linear molecules are lacking or inactive.     

Construction of a plasmid containing functional Escherichia coli uvrA, B, and C genes in a configuration potentially suitable for mammalian expression

A plasmid, pUVABC-2, was constructed that encodes functional uvrA, B, and C genes of Escherichia coli. This plasmid also contains the gpt and ampr genes for positive selection in either bacterial or mammalian systems. Each of the uvrA, B, C, and gpt genes is located between SV40 initiation and termination signals and retains the original bacterial promoters. This recombinant vector conferred a wild-type UV resistance phenotype to uvrA-, B-, and C- strains of E. coli. The results indicate that each of the uvr genes contained in pUVABC-2 function in E. coli. The plasmid is a potential biological probe for DNA repair in mammalian cells.