Caenorhabditis elegans msh-5 is required for both normal and radiation-induced meiotic crossing over but not for completion of meiosis.

Crossing over and chiasma formation during Caenorhabditis elegans meiosis require msh-5, which encodes a conserved germline-specific MutS family member. msh-5 mutant oocytes lack chiasmata between homologous chromosomes, and crossover frequencies are severely reduced in both oocyte and spermatocyte meiosis. Artificially induced DNA breaks do not bypass the requirement for msh-5, suggesting that msh-5 functions after the initiation step of meiotic recombination. msh-5 mutants are apparently competent to repair breaks induced during meiosis, but accomplish repair in a way that does not lead to crossovers between homologs. These results combine with data from budding yeast to establish a conserved role for Msh5 proteins in promoting the crossover outcome of meiotic recombination events. Apart from the crossover deficit, progression through meiotic prophase is largely unperturbed in msh-5 mutants. Homologous chromosomes are fully aligned at the pachytene stage, and germ cells survive to complete meiosis and gametogenesis with high efficiency. Our demonstration that artificially induced breaks generate crossovers and chiasmata using the normal meiotic recombination machinery suggests (1) that association of breaks with a preinitiation complex is not a prerequisite for entering the meiotic recombination pathway and (2) that the decision for a subset of recombination events to become crossovers is made after the initiation step.

The presence of only one of five exoribonucleases is sufficient to support the growth of Escherichia coli.

Escherichia coli contains multiple exoribonucleases. Strains lacking the exoribonucleases RNase II, D, BN, T, and PH are inviable. The introduction of a chromosomal, wild-type copy of the gene for any one of these enzymes is sufficient to allow cell growth, with the enzymes being in the following order of effectiveness: RNase T > RNase PH > RNase D > RNase II > RNase BN. The data indicate that these five exoribonucleases functionally overlap in vivo and that any one of them can take over the functions of all the others, although with various efficiencies.

RNase PH is essential for tRNA processing and viability in RNase-deficient Escherichia coli cells.

RNase PH is a Pi-dependent exoribonuclease that can act at the 3' terminus of tRNA precursors in vitro. To obtain information about the function of this enzyme in vivo, the Escherichia coli rph gene encoding RNase PH was interrupted with either a kanamycin resistance or a chloramphenicol resistance cassette and transferred to the chromosome of a variety of RNase-resistant strains. Inactivation of the chromosomal copy of rph eliminated RNase PH activity from extracts and also slowed the growth of many of the strains, particularly ones that already were deficient in RNase T or polynucleotide phosphorylase. Introduction of the rph mutation into a strain already lacking RNases I, II, D, BN, and T resulted in inviability. The rph mutation also had dramatic effects on tRNA metabolism. Using an in vivo suppressor assay we found that elimination of RNase PH greatly decreased the level of su3+ activity in cells deficient in certain of the other RNases. Moreover, in an in vitro tRNA processing system the defect caused by elimination of RNase PH was shown to be the accumulation of a precursor that contained 4-6 additional 3' nucleotides following the -CCA sequence. These data indicate that RNase PH can be an essential enzyme for the processing of tRNA precursors.

Characterization of Escherichia coli RNase PH.

We have previously shown that the orfE gene of Escherichia coli encodes RNase PH. Here we show that the OrfE protein (purified as described in the accompanying paper) (Jensen, K. F., Andersen, J. T., and Poulsen, P. (1992) J. Biol. Chem. 267, 17147-17152) has both the degradative and synthetic activities of RNase PH. This highly purified protein was used to characterize the enzymatic and structural properties of RNase PH. The enzyme requires a divalent cation and phosphate for activity, the latter property indicating that RNase PH is exclusively a phosphorolytic enzyme. Among tRNA-type substrates, the enzyme is most active against synthetic tRNA precursors containing extra residues following the -CCA sequence, and it can act on these molecules to generate mature tRNA with amino acid acceptor activity; 3'-phosphoryl-terminated molecules are not active as substrates. The equilibrium constant for RNase PH is near unity, suggesting that at the phosphate concentration present in vivo, the enzyme would participate in RNA degradation. The synthetic reaction of RNase PH displays a nonlinear response to increasing enzyme concentrations, and this may be due to self-aggregation of the protein. Higher order multimers of RNase PH could be detected by gel filtration at higher protein concentrations and by protein cross-linking. The possible role of RNase PH in tRNA processing is discussed.