Strains of Escherichia coli with a mutation in the sof(dnaS) locus show a higher than normal frequency of recombination (are hyper rec) and incorporate label into short (4-5S) DNA (Fig. 1). Second, an excision-repair system detects and removes uracil residues that may have escaped the action of dUTPase and were misincorporated into DNA. Lindahl (4) has described an N-glycosidase that catalyzes the cleavage of the uracildeoxyribose linkage in DNA, and nucleases, acting at the apyrinidinic acid site, might excise that region of the backbone (5, 6); the gap could be filled in by DNA polymerase I and DNA ligase to complete the repair process (7). Gates and Linn § have very recently identified an endonuclease that may also serve in removal of uracil residues by its specific capacity to hydrolyze uracil-containing DNA.A defect in dUTPase would be expected to produce an increase in the intracellular pool of dUTP, and in addition, to block the predominant pathway of thymidine nucleotide biosynthesis, both of which should lead to an increased level of uracil in DNA. However, a group of dUTPase mutants recently isolated by Hochhauser and Weiss (8)

We have determined the structure of the homotrimeric dUTPase from Escherichia coli, completed with an inhibitor and substrate analogue, dUDP. Three molecules of dUDP are found symmetrically bound per trimer, each in a shallow cleft between adjacent subunits, interacting with evolutionary conserved residues. The interactions of the uracil ring and the deoxypentose with the protein are consistent with the high specificity of the enzyme with respect to these groups. The positions of the two phosphate groups and adjacent water molecules are discussed in relation to the mechanism and kinetics of catalysis. The role that dUTPase plays in DNA metabolism makes the enzyme a potential target for chemotherapeutic drugs: the results presented here will aid in the design and development of inhibitory compounds.

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In oxygenic photosynthetic organisms the PSI-C polypeptide, encoded by the psaC gene, provides the ligands for two centers, FA and FB, the terminal electron acceptors in the photosystem I (PSI) complex. An insertion mutation introduced in the psaC locus of the filamentous cyanobacteriumAnabaena variabils ATCC 29413 resulted in the creation of a mutant strain, T398-1, that lacks the PSI-C polypeptide. In medium supplemented with 5 mM fructose, the mutant cells grew well in the dark. However, when grown in the same medium under light, the doubling rate of T398-1 cells was significantly decreased. In intact cells of T398-1, bicarbonatedependent whole-chain electron transport (PSII and PSI) could not be detected, although partial electron transport reactions involving either one of the two photosystems could be measured at significant rates. The low-temperature EPR signals attributed to the [4Fe-4S] centers FA and FB were absent in the mutant cells. Chemical titration measurements indicated that the ratios of chlorophyll to the primary donor P700 were virtually identical in membranes from the wild-type and mutant cells. Moreover, room-temperature optical spectroscopic analysis of the thylakoid membranes isolated from T398-1 showed flash-induced P700 oxidation followed by dark rereduction, indicating primary photochemistry in PSI. Thus stable assembly of the reaction center of PSI can occur in the absence of the Fe-S cluster cofactors FA and FB. These studies demonstrate thatAnabaena 29413 offers a useful genetic system for targeted mutagenesis of the PSI complex.Photosystem I (PSI) is a membrane-bound pigment-protein complex that mediates electron transfer from reduced plastocyanin and cytochrome c553 to ferredoxin. Biochemical and biophysical analyses of thylakoid membranes and isolated PSI particles from cyanobacteria and higher plants have helped in formulating the functional organization of redox intermediates involved in the transfer of electrons from P700, the reaction-center chlorophyll(s) (Chl) of PSI, to ferredoxin.Five different electron-transfer intermediates-namely, A0, Al, Fx, FA, and FE-Nare known to be involved in electron transfer from P700 to ferredoxin (reviewed in ref.

The dynamics of the C-terminus of the dUTPases from Escherichia coli and equine infectious anaemia virus (EIAV) were studied by 1 H^1 5 N nuclear magnetic resonance spectroscopy. The two enzymes differ with regard to flexibility in the backbone of the 15 most C-terminal amino acid residues, some of which are conserved and essential for enzymic activity. In the bacterial enzyme, the residues closest to the C-terminus are highly flexible and display a correlation time in the nanosecond time range. No similar high flexibility could be detected for the C-terminal part of EIAV dUTPase, indicating a different time range of flexibility. ß