Lack of functional alpha-lactalbumin prevents involution in Cape fur seals and identifies the protein as an apoptotic milk factor in mammary gland involution

The mammary gland undergoes a sophisticated programme of developmental changes during pregnancy/lactation. However, little is known about processes involving initiation of apoptosis at involution following weaning. We used fur seals as models to study the molecular process of involution as these animals display a unique mammary gland phenotype. Fur seals have long lactation periods whereby mothers cycle between secreting copious quantities of milk for 2 to 3 days suckling pups on land, with trips to sea alone to forage for up to 23 days during which time mammary glands remain active without initiating apoptosis/involution.<br /

DNA origami-based single-molecule forcespectroscopy elucidates RNA Polymerase IIIpre-initiation complex stability

The TATA-binding protein (TBP) and a transcription factor (TF) IIB-like factor are important constituents of all eukaryotic initiation complexes. The reason for the emergence and strict requirement of the additional initiation factor Bdp1 in the RNA polymerase (RNAP) III system, however, remained elusive. A poorly studied aspect in this context is the effect of DNA strain arising from DNA compaction and transcriptional activity on initiation complex formation. We made use of a DNA origami-based force clamp to follow the assembly of human initiation complexes in the RNAP II and RNAP III systems at the single-molecule level under piconewton forces. We demonstrate that TBP-DNA complexes are force-sensitive and TFIIB is sufficient to stabilise TBP on a strained promoter. In contrast, Bdp1 is the pivotal component that ensures stable anchoring of initiation factors, and thus the polymerase itself, in the RNAP III system. Thereby, we offer an explanation for the crucial role of Bdp1 for the high transcriptional output of RNAP III

Yeast replication factor-A functions in the unwinding of the SV40 origin of DNA replication

Cell-free replication systems for simian virus 40 (SV40) DNA are taken to be a model for the replication of eukaryotic chromosomes, because only one viral protein is required to supplement the replication proteins provided by a human cell extract. To prove that these cellular proteins function in chromosomal DNA replication we have begun to identify homologous proteins in an organism that can be genetically manipulated. Here we report the identification of yeast replication factor-A (yRF-A) from Saccharomyces cerevisiae and show that it is functionally and structurally related to a human protein that is required for the initiation and elongation of SV40 DNA replication. Yeast RF-A, a multi-subunit phosphoprotein, is similar to the human protein in its chromatographic behaviour, subunit structure and DNA-binding activity. The yeast protein will fully substitute for the human protein in an early stage of the initiation of SV40 DNA replication. Substitution of yRF-A in the complete SV40 replication system, however, results in reduced DNA replication, presumably due to a requirement for species-specific interactions between yeast RF-A and the DNA polymerase complex

Kinetic analysis of yersinia pestis DNA adenine methyltransferase activity using a hemimethylated molecular break light oligonucleotide

Background: DNA adenine methylation plays an important role in several critical bacterial processes including mismatchrepair, the timing of DNA replication and the transcriptional control of gene expression. The dependence of bacterial virulenceon DNA adenine methyltransferase (Dam) has led to the proposal that selective Dam inhibitors might function as broadspectrum antibiotics. Methodology/Principal Findings: herein we report the expression and purification of Yersinia pestisDam and the development of a continuous fluorescence based assay for DNA adenine methyltransferase activity that issuitable for determining the kinetic parameters of the enzyme and for high throughput screening against potential Daminhibitors. The assay utilised a hemimethylated break light oligonucleotide substrate containing a GATC methylation site.When this substrate was fully methylated by Dam, it became a substrate for the restriction enzyme DpnI, resulting inseparation of fluorophore (fluorescein) and quencher (dabcyl) and therefore an increase in fluorescence. The assays weremonitored in real time using a fluorescence microplate reader in 96 well format and were used for the kinetic characterisationof Yersinia pestis Dam, its substrates and the known Dam inhibitor, S-adenosylhomocysteine. The assay has been validated forhigh throughput screening, giving a Z-factor of 0.7160.07 indicating that it is a sensitive assay for the identification ofinhibitors. Conclusions/Significance: the assay is therefore suitable for high throughput screening for inhibitors of DNAadenine methyltransferases and the kinetic characterisation of the inhibitio