Helmut Pospiech

Detection of Intracellular Bacteria in the Buds of Scotch Pine (Pinus sylvestris L.) by In Situ Hybridization

ABSTRACT Bacterial isolates were obtained from pine (Pinus sylvestris L.) tissue cultures and identified asMethylobacterium extorquens and Pseudomonas synxantha. The existence of bacteria in pine buds was investigated by 16S rRNA in situ hybridization. Bacteria inhabited the buds of every tree examined, primarily colonizing the cells of scale primordia and resin ducts.

A novel family of DNA-polymerase-associated B subunits

This work was supported by a grant from the Research Council for the Environment and Natural Resources, Academy of Finland.

Characterization of cDNAs for mouse lysyl hydroxylase 1, 2 and 3, their phylogenetic analysis and tissue-specific expression in the mouse

We report on the isolation and characterization of cDNA clones for mouse lysyl hydroxylases 1, 2 and 3 (LH1, LH2, LH3). Phylogenetic analysis using nine lysyl hydroxylase sequences from five species indicates that the isoforms are derived from an ancestral gene by two duplication events, isoforms 1 and 2 being more closely related and having resulted from a more recent duplication than isoform 3. Expression of the isoforms is highly regulated in adult mouse tissues. LH1 is strongly expressed in the liver, heart, lung, skeletal muscle and kidney tissue, LH2 expression is high in the heart, lung, kidney, eye, ovary and placenta, whereas LH3 expression is high in the heart, lung, liver and testis tissue.

Two endophytic fungi in different tissues of scots pine buds (Pinus sylvestris L.).

Two fungal species were isolated with different frequencies from pine tissue cultures originating from buds. One species was detected in 33.1% of the cultures initiated in March, and another was present in 1.7% of cultures initiated in June. Based on analyses of phylogenetic and physiological characteristics these fungi were identified as Hormonema dematioides (isolated in March) and Rhodotorula minuta (isolated in June). Probes targeted towards the 18S rRNA of H. dematioides and R. minuta were made. When in situ hybridizations were performed on pine bud tissue, R. minuta was detected inside the cells of meristematic tissue in 40% of the samples, in contrast to H. dematioides, which was not found in this tissue. Using light microscopy, H. dematioides was found to be localized in the scale tissues of the buds. Fungal endophytes have previously been detected in scale tissues, but not in the meristematic tissues of buds. The habitats of these fungi may reflect their different roles in the plant.

An ancient genetic link between vertebrate mitochondrial fatty acid synthesis and RNA processing

In bacteria, functionally related gene products are often encoded by a common transcript. Such polycistronic transcripts are rare in eukaryotes. Here we isolated several clones from human cDNA libraries, which rescued the respiratory‐deficient phe‐notype of a yeast mitochondrial 3‐hydroxyacyl thioester dehydratase 2 (htd2) mutant strain. All complementing cDNAs were derived from the RPP14 transcript previously described to encode the RPP14 subunit of the human ribonuclease P (RNase P) complex. We identified a second, 3′ open reading frame (ORF) on the RPP14 transcript encoding a protein showing similarity to known dehydratases and hydratase 2 enzymes. The protein was localized in mitochondria, and the recombinant enzyme exhibited (3R)‐specific hydratase 2 activity. Based on our results, we named the protein human 3‐hydroxyacyl‐thioester dehydratase 2 (HsHTD2), which is involved in mitochondrial fatty acid synthesis. The bicistronic arrangement of RPP14 and HsHTD2, as well as the general exon structure of the gene, is conserved in vertebrates from fish to humans, indicating a genetic link conserved for 400 million years between RNA processing and mitochondrial fatty acid synthesis.—Autio, K. J., Kastaniotis, A. J., Pospiech, H., Miinalainen, I. J., Schonauer, M. S., Dieckmann, C. L., Hiltunen, J. K. An ancient genetic link between vertebrate mitochondrial fatty acid synthesis and RNA processing. FASEB J. 22, 569–578 (2008)

DNA Polymerase e - More Than a Polymerase

This paper presents a comprehensive review of the structure and function of DNA polymerase e. Together with DNA polymerases a and d, this enzyme replicates the nuclear DNA in the eukaryotic cell. During this process, DNA polymerase a lays down RNA-DNA primers that are utilized by DNA polymerases d and e for the bulk DNA synthesis. Attempts have been made to assign these two enzymes specifically to the synthesis of the leading and the lagging strand. Alternatively, the two DNA polymerases may be needed to replicate distinct regions depending on chromatin structure. Surprisingly, the essential function of DNA polymerase e does not depend on its catalytic activity, but resides in the nonenzymatic carboxy-terminal domain. This domain not only mediates the interaction of the catalytic subunit with the three smaller regulatory subunits, but also links the replication machinery to the S phase checkpoint. In addition to its role in DNA replication, DNA polymerase e fulfils roles in the DNA synthesis step of nucleotide excision and base excision repair, and has been implicated in recombinational processes in the cell.

Heterozygous mutations in PALB2 cause DNA replication and damage response defects

Besides mutations in BRCA1/BRCA2, heterozygous defects in PALB2 are important in breast cancer predisposition. PALB2 heterozygosity increases the risk of malignancy about sixfold. PALB2 interacts with BRCA1 and BRCA2 to regulate homologous recombination and mediate DNA damage response. Here we show, by analysing lymphoblastoid cell lines from heterozygous female PALB2 mutation carriers, that PALB2 haploinsufficiency causes aberrant DNA replication/damage response. Mutation carrier cells show increased origin firing and shorter distance between consecutive replication forks. Carrier cell lines also show elevated ATR protein, but not phosphorylation levels, and a majority of them display aberrant Chk1-/Chk2-mediated DNA damage response. Elevated chromosome instability is observed in primary blood lymphocytes of PALB2 mutation carriers, indicating that the described mechanisms of genome destabilization operate also at the organism level. These findings provide a new mechanism for early stages of breast cancer development that may also apply to other heterozygous homologous recombination signalling pathway gene mutations in hereditary cancer predisposition.

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.

The glycosyltransferase activities of lysyl hydroxylase 3 (LH3) in the extracellular space are important for cell growth and viability

Lysyl hydroxylase (LH) isoform 3 is a post‐translational enzyme possessing LH, collagen galactosyltransferase (GT) and glucosyltransferase (GGT) activities. We have demonstrated that LH3 is found not only intracellularly, but also on the cell surface and in the extracellular space, suggesting additional functions for LH3. Here we show that the targeted disruption of LH3 by siRNA causes a marked reduction of both glycosyltransferase activities, and the overexpression of LH3 in HT‐1080 cells increases hydroxylation of lysyl residues and the subsequent galactosylation and glucosylation of hydroxylysyl residues. These data confirm the multi‐functionality of LH3 in cells. Furthermore, treatment of cells in culture medium with a LH3 N‐terminal fragment affects the cell behaviour, rapidly leading to arrest of growth and further to lethality if the fragment is glycosyltransferase‐deficient, and leading to stimulation of proliferation if the fragment contains LH3 glycosyltransferase activities. The effect is reversible, the cells recovering after removal of the glycosyltransferase‐deficient fragment. The findings were confirmed by overexpressing the full‐length LH3 in native or mutated forms in the cells. The data indicate that the increase in proliferation depends on the glycosyltransferase activity of LH3. The overexpression of a glycosyltransferase‐deficient mutant or targeted disruption of LH3 by siRNA in cells results in abnormal cell morphology followed by cell death. Our data clearly indicate that the deficiency of LH3 glycosyltransferase activities, especially in the extracellular space, causes growth arrest revealing the importance of the glycosyltransferase activities of LH3 for cell growth and viability, and identifying LH3 as a potential target for medical applications, such as cancer therapy.

The intrinsically disordered amino-terminal region of human RecQL4: multiple DNA-binding domains confer annealing, strand exchange and G4 DNA binding

Human RecQL4 belongs to the ubiquitous RecQ helicase family. Its N-terminal region represents the only homologue of the essential DNA replication initiation factor Sld2 of Saccharomyces cerevisiae, and also participates in the vertebrate initiation of DNA replication. Here, we utilized a random screen to identify N-terminal fragments of human RecQL4 that could be stably expressed in and purified from Escherichia coli. Biophysical characterization of these fragments revealed that the Sld2 homologous RecQL4 N-terminal domain carries large intrinsically disordered regions. The N-terminal fragments were sufficient for the strong annealing activity of RecQL4. Moreover, this activity appeared to be the basis for an ATP-independent strand exchange activity. Both activities relied on multiple DNA-binding sites with affinities to single-stranded, double-stranded and Y-structured DNA. Finally, we found a remarkable affinity of the N-terminus for guanine quadruplex (G4) DNA, exceeding the affinities for other DNA structures by at least 60-fold. Together, these findings suggest that the DNA interactions mediated by the N-terminal region of human RecQL4 represent a central function at the replication fork. The presented data may also provide a mechanistic explanation for the role of elements with a G4-forming propensity identified in the vicinity of vertebrate origins of DNA replication.