Gabriel Kaufmann

Bacteriophage T4 anticodon nuclease, polynucleotide kinase and RNA ligase reprocess the host lysine tRNA.

Host tRNAs cleaved near the anticodon occur specifically in T4‐infected Escherichia coli prr strains which restrict polynucleotide kinase (pnk) or RNA ligase (rli) phage mutants. The cleavage products are transient with wt but accumulate in pnk‐ or rli‐ infections, implicating the affected enzymes in repair of the damaged tRNAs. Their roles in the pathway were elucidated by comparing the mutant infection intermediates with intact tRNA counterparts before or late in wt infection. Thus, the T4‐induced anticodon nuclease cleaves lysine tRNA 5′ to the wobble position, yielding 2′:3′‐P greater than and 5′‐OH termini. Polynucleotide kinase converts them into a 3′‐OH and 5′ P pair joined in turn by RNA ligase. Presumably, lysine tRNA depletion, in the absence of polynucleotide kinase and RNA ligase mediated repair, underlies prr restriction. However, the nuclease, kinase and ligase may benefit T4 directly, by adapting levels or decoding specificities of host tRNAs to T4 codon usage.

The optional E. coli prr locus encodes a latent form of phage T4-induced anticodon nuclease.

The optional Escherichia coli prr locus restricts phage T4 mutants lacking polynucleotide kinase or RNA ligase. Underlying this restriction is the specific manifestation of the T4‐induced anticodon nuclease, an enzyme which triggers the cleavage‐ligation of the host tRNALys. We report here the molecular cloning, nucleotide sequence and mutational analysis of prr‐associated DNA. The results indicate that prr encodes a latent form of anticodon nuclease consisting of a core enzyme and cognate masking agents. They suggest that the T4‐encoded factors of anticodon nuclease counteract the prr‐encoded masking agents, thus activating the latent enzyme. The encoding of a tRNA cleavage‐ligation pathway by two separate genetic systems which cohabitate E. coli may provide a clue to the evolution of RNA splicing mechanisms mediated by proteins.

Cloning of nascent monkey DNA synthesized early in the cell cycle.

To study the structure and complexity of animal cell replication origins, we have isolated and cloned nascent DNA from the onset of S phase as follows: African green monkey kidney cells arrested in G1 phase were serum stimulated in the presence of the DNA replication inhibitor aphidicolin. After 18 h, the drug was removed, and DNA synthesis was allowed to proceed in vivo for 1 min. Nuclei were then prepared, and DNA synthesis was briefly continued in the presence of Hg-dCTP. The mercury-labeled nascent DNA was purified in double-stranded form by extrusion (M. Zannis-Hadjopoulos, M. Perisco, and R. G. Martin, Cell 27:155-163, 1981) followed by sulfhydryl-agarose affinity chromatography. Purified nascent DNA (ca. 500 to 2,000 base pairs) was treated with mung bean nuclease to remove single-stranded ends and inserted into the NruI site of plasmid pBR322. The cloned fragments were examined for their time of replication by hybridization to cellular DNA fractions synthesized at various intervals of the S phase. Among five clones examined, four hybridized preferentially with early replicating fractions.

The Middle Subunit of Replication Protein A Contacts Growing RNA-DNA Primers in Replicating Simian Virus 40 Chromosomes

ABSTRACT The eukaryotic single-stranded DNA binding protein replication protein A (RPA) participates in major DNA transactions. RPA also interacts through its middle subunit (Rpa2) with regulators of the cell division cycle and of the response to DNA damage. A specific contact between Rpa2 and nascent simian virus 40 DNA was revealed by in situ UV cross-linking. The dynamic attributes of the cross-linked DNA, namely, its size distribution, RNA primer content, and replication fork polarity, were determined. These data suggest that Rpa2 contacts the early DNA chain intermediates synthesized by DNA polymerase α-primase (RNA-DNA primers) but not more advanced products. Possible signaling functions of Rpa2 are discussed, and current models of eukaryotic lagging-strand DNA synthesis are evaluated in view of our results.

Properties of some monkey DNA sequences obtained by a procedure that enriches for DNA replication origins.

Twelve clones of monkey DNA obtained by a procedure that enriches 10(3)- to 10(4)-fold for nascent sequences activated early in S phase (G. Kaufmann, M. Zannis-Hadjopoulos, and R. G. Martin, Mol. Cell. Biol. 5:721-727, 1985) have been examined. Only 2 of the 12 ors sequences (origin-enriched sequences) are unique (ors1 and ors8). Three contain the highly reiterated Alu family (ors3, ors9, and ors11). One contains the highly reiterated alpha-satellite family (ors12), but none contain the Kpn family. Those remaining contain middle repetitive sequences. Two examples of the same middle repetitive sequence were found (ors2 and ors6). Three of the middle repetitive sequences (the ors2-ors6 pair, ors5, and ors10) are moderately dispersed; one (ors4) is highly dispersed. The last, ors7, has been mapped to the bona fide replication origin of the D loop of mitochondrial DNA. Of the nine ors sequences tested, half possess snapback (intrachain reannealing) properties.

T4 RNA ligase: Substrate chain length requirements

RNA ligase was discovered by Silber et al. [ 1 ] in extracts of E. coli cells infected with T4 bacteriophage. The enzyme catalyzes the conversion of linear polyribonucleotides to a circular form. In this reaction an intramolecular covalent linkage is formed between the 5’-phosphate end and the 3’-hydroxyl terminus. Such an intramolecular joining reaction is thought to be constrained by the probabilities of juxtaposition of the reactive termini and by the strain induced upon ring closure [2], Thus, the rate of this reaction is expected to vary with chain length and composition of the reacting polyribonucleotides. Knowledge of these factors may serve as a measure of the conformation of various polyribonucleotides and indicate the in vivo role of RNA ligase. In the present studies we examined a series of oligo (PA) with increasing chain length as substrates for the enzyme. It was found that (pA)s is the shortest oligonucleotide in this series that can be circularized by the enzyme.

Distinctive activities of DNA polymerases during human DNA replication

The contributions of human DNA polymerases (pols) α, δ and ε during S‐phase progression were studied in order to elaborate how these enzymes co‐ordinate their functions during nuclear DNA replication. Pol δ was three to four times more intensely UV cross‐linked to nascent DNA in late compared with early S phase, whereas the cross‐linking of pols α and ε remained nearly constant throughout the S phase. Consistently, the chromatin‐bound fraction of pol δ, unlike pols α and ε, increased in the late S phase. Moreover, pol δ neutralizing antibodies inhibited replicative DNA synthesis most efficiently in late S‐phase nuclei, whereas antibodies against pol ε were most potent in early S phase. Ultrastructural localization of the pols by immuno‐electron microscopy revealed pol ε to localize predominantly to ring‐shaped clusters at electron‐dense regions of the nucleus, whereas pol δ was mainly dispersed on fibrous structures. Pol α and proliferating cell nuclear antigen displayed partial colocalization with pol δ and ε, despite the very limited colocalization of the latter two pols. These data are consistent with models where pols δ and ε pursue their functions at least partly independently during DNA replication.

RloC: a wobble nucleotide‐excising and zinc‐responsive bacterial tRNase

The conserved bacterial protein RloC, a distant homologue of the tRNALys anticodon nuclease (ACNase) PrrC, is shown here to act as a wobble nucleotide‐excising and Zn++‐responsive tRNase. The more familiar PrrC is silenced by a genetically linked type I DNA restriction‐modification (R‐M) enzyme, activated by a phage anti‐DNA restriction factor and counteracted by phage tRNA repair enzymes. RloC shares PrrCs ABC ATPase motifs and catalytic ACNase triad but features a distinct zinc‐hook/coiled‐coil insert that renders its ATPase domain similar to Rad50 and related DNA repair proteins. Geobacillus kaustophilus RloC expressed in Escherichia coli exhibited ACNase activity that differed from PrrCs in substrate preference and ability to excise the wobble nucleotide. The latter specificity could impede reversal by phage tRNA repair enzymes and account perhaps for RloCs more frequent occurrence. Mutagenesis and functional assays confirmed RloCs catalytic triad assignment and implicated its zinc hook in regulating the ACNase function. Unlike PrrC, RloC is rarely linked to a type I R‐M system but other genomic attributes suggest their possible interaction in trans. As DNA damage alleviates type I DNA restriction, we further propose that these related perturbations prompt RloC to disable translation and thus ward off phage escaping DNA restriction during the recovery from DNA damage.

HSD restriction-modification proteins partake in latent anticodon nuclease.

Phage T4‐induced anticodon nuclease triggers cleavage‐ligation of the host tRNA(Lys). The enzyme is encoded in latent form by the optional Escherichia coli locus prr and is activated by the product of the phage stp gene. Anticodon nuclease latency is attributed to the masking of the core function prrC by flanking elements homologous with type I restriction‐modification genes (prrA‐hsdM and prrD‐hsdR). Activation of anticodon nuclease in extracts of uninfected prr+ cells required synthetic Stp, ATP and GTP and appeared to depend on endogenous DNA. Stp could be substituted by a small, heat‐stable E. coli factor, hinting that anticodon nuclease may be mobilized in cellular situations other than T4 infection. Hsd antibodies recognized the anticodon nuclease holoenzyme but not the prrC‐encoded core. Taken together, these data indicate that Hsd proteins partake in the latent ACNase complex where they mask the core factor PrrC. Presumably, this masking interaction is disrupted by Stp in conjunction with Hsd ligands. The Hsd‐PrrC interaction may signify coupling and mutual enhancement of two prokaryotic restriction systems operating at the DNA and tRNA levels.

Mammalian DNA enriched for replication origins is enriched for snap-back sequences☆

Using the instability of replication loops as a method for the isolation of double-stranded nascent DNA, extruded DNA enriched for replication origins was obtained and denatured. Snap-back DNA, single-stranded DNA with inverted repeats (palindromic sequences), reassociates rapidly into stem-loop structures with zero-order kinetics when conditions are changed from denaturing to renaturing, and can be assayed by chromatography on hydroxyapatite. Origin-enriched nascent DNA strands from mouse, rat and monkey cells growing either synchronously or asynchronously were purified and assayed for the presence of snap-back sequences. The results show that origin-enriched DNA is also enriched for snap-back sequences, implying that some origins for mammalian DNA replication contain or lie near palindromic sequences.