DNA double-strand break-induced phosphorylation of Drosophila histone variant H2Av helps prevent radiation-induced apoptosis

The response of eukaryotic cells to the formation of a double-strand break (DSB) in chromosomal DNA is highly conserved. One of the earliest responses to DSB formation is phosphorylation of the C-terminal tail of H2A histones located in nucleosomes near the break. Histone variant H2AX and core histone H2A are phosphorylated in mammals and budding yeast, respectively. We demonstrate the DSB-induced phosphorylation of histone variant H2Av in Drosophila melanogaster. H2Av is a member of the H2AZ family of histone variants. Ser137 within an SQ motif located near the C- terminus of H2Av was phosphorylated in response to γ-irradiation in both tissue culture cells and larvae. Phosphorylation was detected within 1 min of irradiation and detectable after only 0.3 Gy of radiation exposure. Photochemically induced DSBs, but not general oxidative damage or UV-induced nicking of DNA, caused H2Av phosphorylation, suggesting that phosphorylation is DSB specific. Imaginal disc cells from Drosophila expressing a mutant allele of H2Av with its C-terminal tail deleted, and therefore unable to be phosphorylated, were more sensitive to radiation-induced apoptosis than were wildtype controls, suggesting that phosphorylation of H2Av is important for repair of radiation-induced DSBs. These observations suggest that in addition to providing the function of an H2AZ histone, H2Av is also the functional homolog in Drosophila of H2AX.     

Mitochondrial Iba57p is required for Fe/S cluster formation on aconitase and activation of radical SAM enzymes

A genome-wide screen for Saccharomyces cerevisiae iron-sulfur (Fe/S) cluster assembly mutants identified the gene IBA57. The encoded protein Iba57p is located in the mitochondrial matrix and is essential for mitochondrial DNA maintenance. The growth phenotypes of an iba57Δ mutant and extensive functional studies in vivo and in vitro indicate a specific role for Iba57p in the maturation of mitochondrial aconitase-type and radical SAM Fe/S proteins (biotin and lipoic acid synthases). Maturation of other Fe/S proteins occurred normally in the absence of Iba57p. These observations identify Iba57p as a novel dedicated maturation factor with specificity for a subset of Fe/S proteins. The Iba57p primary sequence is distinct from any known Fe/S assembly factor but is similar to certain tetrahydrofolate-binding enzymes, adding a surprising new function to this protein family. Iba57p physically interacts with the mitochondrial ISC assembly components Isa1p and Isa2p. Since all three proteins are conserved in eukaryotes and bacteria, the specificity of the Iba57/Isa complex may represent a biosynthetic concept that is universally used in nature. In keeping with this idea, the human IBA57 homolog C1orf69 complements the iba57Δ growth defects, demonstrating its conserved function throughout the eukaryotic kingdom.     

Structural and Functional Studies on the Overproduced L11 Protein from Thermus thermophilus

The L11 ribosomal protein from Thermus thermophilus (TthL11) has been overproduced and purified to homogeneity using a two-step purification protocol. The overproduced protein carries a similar methylation pattern at Lys-3 as does its homolog from Escherichia coli. Chymotrypsin digested only a small part of the TthL11 protein and did not cleave TthL11 into two peptides, as in the case of EcoL11, but produced only a single N-terminal peptide. Tryptic digestion of TthL11 also produced an N-terminal peptide, in contrast to the C-terminal peptide obtained with L11 from Bacillus stearothermophilus. The recombinant protein forms a specific complex with a 55-nt 23S rRNA fragment known to interact with members of the L11 family from several organisms. Cooperative binding of TthL11 and thiostrepton to 23S rRNA leads to an increased protection of TthL11 from tryptic digestion. The similar structural and biochemical properties as well as the significant homology between L11 from E. coli and B. stearothermophilus with the corresponding protein from Thermus thermophilus indicate an evolutionarily conserved protein important for ribosome function.     

Characterization of a novel small RNA encoded by Dictyostelium discoideum mitochondrial DNA

In this study, we analyzed a mitochondrial small (ms) RNA in Dictyostelium discoideum, which is 129 nucleotides long and has a GC content of only 22.5%. In the mitochondrial DNA, a single-copy gene (msr) for the ms RNA was located downstream of the gene for large-subunit rRNA. The location of msr was similar to that of the 5S rRNA gene in prokaryotes and chloroplasts, but clearly different from that in mitochondria of plants, liverwort and the chlorophycean alga Prototheca wikerhamii, in which small-subunit rRNA and 5S rRNA genes are closely linked. The primary sequence of ms RNA showed low homology with mitochondrial 5S rRNA from plants, liverwort and the chlorophycean alga, but the proposed secondary structure of ms RNA was similar to that of cytoplasmic 5S rRNA. In addition, ms RNA showed a highly conserved GAAC sequence in the same loop as in common 5S rRNA. However, ms RNA was detected mainly in the mitochondrial 25?000?×?g supernatant fraction which was devoid of ribosomes. It is possible that ms RNA is an evolutionary derivative of mitochondrial 5S rRNA.     

Dimerization Mediated by a Divergent Forkhead-associated Domain Is Essential for the DNA Damage and Spindle Functions of Fission Yeast Mdb1

MDC1 is a key factor of DNA damage response in mammalian cells. It possesses two phospho-binding domains. In its C terminus, a tandem BRCA1 C-terminal domain binds phosphorylated histone H2AX, and in its N terminus, a forkhead-associated (FHA) domain mediates a phosphorylation-enhanced homodimerization. The FHA domain of the Drosophila homolog of MDC1, MU2, also forms a homodimer but utilizes a different dimer interface. The functional importance of the dimerization of MDC1 family proteins is uncertain. In the fission yeast Schizosaccharomyces pombe, a protein sharing homology with MDC1 in the tandem BRCA1 C-terminal domain, Mdb1, regulates DNA damage response and mitotic spindle functions. Here, we report the crystal structure of the N-terminal 91 amino acids of Mdb1. Despite a lack of obvious sequence conservation to the FHA domain of MDC1, this region of Mdb1 adopts an FHA-like fold and is therefore termed Mdb1-FHA. Unlike canonical FHA domains, Mdb1-FHA lacks all the conserved phospho-binding residues. It forms a stable homodimer through an interface distinct from those of MDC1 and MU2. Mdb1-FHA is important for the localization of Mdb1 to DNA damage sites and the spindle midzone, contributes to the roles of Mdb1 in cellular responses to genotoxins and an antimicrotubule drug, and promotes in vitro binding of Mdb1 to a phospho-H2A peptide. The defects caused by the loss of Mdb1-FHA can be rescued by fusion with either of two heterologous dimerization domains, suggesting that the main function of Mdb1-FHA is mediating dimerization. Our data support that FHA-mediated dimerization is conserved for MDC1 family proteins.     

Phage Orf Family Recombinases: Conservation of Activities and Involvement of the Central Channel in DNA Binding

Genetic and biochemical evidence suggests that λ Orf is a recombination mediator, promoting nucleation of either bacterial RecA or phage Redβ recombinases onto single-stranded DNA (ssDNA) bound by SSB protein. We have identified a diverse family of Orf proteins that includes representatives implicated in DNA base flipping and those fused to an HNH endonuclease domain. To confirm a functional relationship with the Orf family, a distantly-related homolog, YbcN, from Escherichia coli cryptic prophage DLP12 was purified and characterized. As with its λ relative, YbcN showed a preference for binding ssDNA over duplex. Neither Orf nor YbcN displayed a significant preference for duplex DNA containing mismatches or 1-3 nucleotide bulges. YbcN also bound E. coli SSB, although unlike Orf, it failed to associate with an SSB mutant lacking the flexible C-terminal tail involved in coordinating heterologous protein-protein interactions. Residues conserved in the Orf family that flank the central cavity in the λ Orf crystal structure were targeted for mutagenesis to help determine the mode of DNA binding. Several of these mutant proteins showed significant defects in DNA binding consistent with the central aperture being important for substrate recognition. The widespread conservation of Orf-like proteins highlights the importance of targeting SSB coated ssDNA during lambdoid phage recombination.