RecQ helicases are essential for the maintenance of chromosome stability. In addition to DNA unwinding, some RecQ enzymes have an intrinsic DNA strand annealing activity. The function of this dual enzymatic activity and the mechanism that regulates it is, however, unknown. Here, we describe two quaternary forms of the human RECQ1 helicase, higher-order oligomers consistent with pentamers or hexamers, and smaller oligomers consistent with monomers or dimers. Size exclusion chromatography and transmission electron microscopy show that the equilibrium between the two assembly states is affected by single-stranded DNA (ssDNA) and ATP binding, where ATP or ATPγS favors the smaller oligomeric form. Our three-dimensional electron microscopy reconstructions of human RECQ1 reveal a complex cage-like structure of approximately 120 Å × 130 Å with a central pore. This oligomeric structure is stabilized under conditions in which RECQ1 is proficient in strand annealing. In contrast, competition experiments with the ATPase-deficient K119R and E220Q mutants indicate that RECQ1 monomers, or tight binding dimers, are required for DNA unwinding. Collectively, our findings suggest that higher-order oligomers are associated with DNA strand annealing, and lower-order oligomers with DNA unwinding.

Human brain astrocytomas range from the indolent low-grade to the highly infiltrating and aggressive high-grade form, also known as glioblastoma multiforme. The extensive heterogeneity of astrocytic tumors complicates their pathological classification. In this study, we compared the protein pattern of low-grade fibrillary astrocytomas to that of glioblastoma multiforme by 2D electrophoresis. The level of most proteins remains unchanged between the different grade tumors and only few differences are reproducibly observable. Fifteen differentially expressed proteins, as well as seventy conserved spots, were identified by mass spectrometry. Western and immnunohistochemical analysis confirmed the differential expression of the identified proteins. These data provide an initial reference map for brain gliomas. Among the proteins more highly expressed in glioblastoma multiforme, we found peroxiredoxin 1 and 6, the transcription factor BTF3, and alpha-B-crystallin, whereas protein disulfide isomerase A3, the catalytic subunit of the cAMP-dependent protein kinase, and the glial fibrillary acidic protein are increased in low-grade astrocytomas. Our findings contribute to deepening our knowledge of the factors that characterize this class of tumors and, at the same time, can be applied toward the development of novel molecular biomakers potentially useful for an accurate classification of the grade of astrocytomas.

The development of this simple treatment allowed the analysis of longer fragments of DNA obtained from archival postmortem paraffin wax embedded tissues.

No abstract

No abstract