CDK-dependent phosphorylation of BRCA2 as a regulatory mechanism for recombinational repair

Inherited mutations in BRCA2 are associated with a predisposition to early-onset breast cancers. The underlying basis of tumorigenesis is thought to be linked to defects in DNA double-strand break repair by homologous recombination. Here we show that the carboxy-terminal region of BRCA2, which interacts directly with the essential recombination protein RAD51, contains a site (serine 3291; S3291) that is phosphorylated by cyclin-dependent kinases. Phosphorylation of S3291 is low in S phase when recombination is active, but increases as cells progress towards mitosis. This modification blocks C-terminal interactions between BRCA2 and RAD51. However, DNA damage overcomes cell cycle regulation by decreasing S3291 phosphorylation and stimulating interactions with RAD51. These results indicate that S3291 phosphorylation might provide a molecular switch to regulate RAD51 recombination activity, providing new insight into why BRCA2 C-terminal deletions lead to radiation sensitivity and cancer predisposition.     

p53 and DNA polymerase alpha compete for binding to SV40 T antigen

The large T antigen (T) of simian virus 40 is a multifunctional protein required for both viral DNA replication and cellular transformation. T antigen forms specific protein complexes with the host protein p53 in both virus-infected and transformed cells. p53 has recently been shown to be an oncogene, but its normal function is not clear. We previously established a radioimmunoassay to study the newly described complex between T antigen and DNA polymerase alpha, and have noted a similarity between the antigenic changes induced in T by the binding of both p53 and polymerase. We now extend this analysis to a larger collection of anti-T antibodies and formally establish that p53 and DNA polymerase alpha can compete for binding to the SV40 T antigen. At a critical concentration of the three components it is possible to detect a trimeric complex of T, p53 and DNA polymerase alpha. Our observations have important implications for the control by these nuclear oncogenes of viral and cellular DNA synthesis and viral host range in both normal and transformed cells. We present a model for the action of p53 in growth control.     

The ovalbumin gene region: common features in the organisation of three genes expressed in chicken oviduct under hormonal control.

Two large DNA fragments overlapping the chicken ovalbumin gene have been isolated by molecular cloning. Analysis of these fragments provided a map of a 46,000-base pair region of the chicken genome. This region contains the complete ovalbumin gene (including its mRNA leader-coding sequence) and at least two other genes of unknown function. All three genes are orientated in the same direction and their expression in chicken oviduct is under hormonal control. The three genes share some sequence homologies, suggesting that duplications have occurred in the ovalbumin gene region in the course of evolution.     

The role of the transcriptional regulator Ptf1a in converting intestinal to pancreatic progenitors

Pancreas development begins with the formation of buds at specific sites in the embryonic foregut endoderm. We used recombination-based lineage tracing in vivo to show that Ptf1a (also known as PTF1-p48) is expressed at these early stages in the progenitors of pancreatic ducts, exocrine and endocrine cells, rather than being an exocrine-specific gene as previously described. Moreover, inactivation of Ptf1a switches the character of pancreatic progenitors such that their progeny proliferate in and adopt the normal fates of duodenal epithelium, including its stem-cell compartment. Consistent with the proposal that Ptf1a supports the specification of precursors of all three pancreatic cell types, transgene-based expression of Pdx1, a gene essential to pancreas formation, from Ptf1a cis-regulatory sequences restores pancreas tissue to Pdx1-null mice that otherwise lack mature exocrine and endocrine cells because of an early arrest in organogenesis. These experiments provide evidence that Ptf1a expression is specifically connected to the acquisition of pancreatic fate by undifferentiated foregut endoderm.     

Protein synthesis required to anchor a mutant p53 protein which is temperature-sensitive for nuclear transport

The p53 protein is rendered temperature-sensitive by a point mutation. Rat cells transformed by this mutant p53 and an activated ras oncogene grow well at 37 degrees C but cease DNA synthesis and cell division when shifted to 32 degrees C. Immunostaining demonstrates that the mutant p53 protein is in the nucleus of the arrested cells at 32 degrees C but in the cytoplasm of the growing cells at 37 degrees C. This is the first example of a protein which is temperature-sensitive for nuclear transport. The translocation from cytoplasm to nucleus and vice versa occurs 6 h after temperature shift and is coincident with the inhibition of DNA synthesis; transport from cytoplasm to nucleus does not require protein synthesis. Remarkably, inhibition of protein synthesis at 37 degrees C also results in the rapid appearance of mutant p53 in the cell nucleus. These results suggest the presence of a short-lived protein responsible for holding p53 in the cytoplasm at 37 degrees C but not at 32 degrees C. Analysis of a non-temperature-sensitive mutant p53 protein shows that its cytoplasmic location is sensitive to protein synthesis inhibitors but not to temperature.