Functional characterization of Pura in vivo and in vitro - Pura knock-out mice and Pura DNA unwinding activity

摘要

Pura is a ubiquitous DNA- and RNA-binding protein that is highly conserved in eukaryoticorganisms. Pura binds preferentially to purine-rich single-stranded DNA. To date,biochemical analyses of Pura have indicated that this protein might be involved in diversecellular functions, including DNA replication, transcription, translation and cell growthcontrol (Gallia et al., 2000). However, the function of Pura in vivo remains unclear.In this study, Pura knock-out mice (KO) were generated using homologous recombinationtechniques in ES cells. The Pura gene locus was isolated from 129/SvJ Mouse GenomicLibrary and partially sequenced. A targeting vector using positive and negative selection wasgenerated for homologous recombination and was finally electroporated into E14 ES cells forfive times. Out of 2400 ES cell clones only one positive ES cell clone (KO 248) wasidentified by Southern-blot analysis.Pura KO mice generated from ES cell clone KO 248 displayed multiple phenotypes.Apparently, they were normal when born, but developed assorted abnormalities. 12-14 daysafter birth they developed a severe and continuous tremor phenotype similar to shiverer micethat lack myelin basic protein (MBP) (Chernoff, 1981). This tremor phenotype continuedunabated until natural death occurred. Pura KO mice seemed to have a shorter life span sincenone of the Pura KO mice lived longer than 6 months while the wild type mice livednormally. Pura has been shown to be involved in developmental and transcriptional control ofMBP (Haas et al., 1995), therefore the MBP levels in the brain stems of Pura KO mice wereinvestigated. It could be shown that MBP levels in 16-day-old Pura KO mice were decreasedby 30-40% as compared to heterozygotes and wild type. However, MBP levels of the 63-dayoldPura KO, heterozygous and wild type mice did not show significant difference. AlthoughPura KO mice showed a tremor phenotype comparable to shiverer mice and displayedreduced MBP levels at the beginning of brain myelination, they still possessed 60-70% ofnormal amounts of MBP, which has been shown to be sufficient to rescue the tremorphenotype in transgenic shiverer mice. These data suggest that the tremor phenotype of PuraKO mice may be due to some other reasons, which are still unclear.Strikingly, 70-day-old Pura KO mice displayed a steadily increasing obesity and significantlyenlarged brain size with increased lipid content, suggesting aberrant fat metabolism orhomeostasis, although the underlying cause(s) are as yet unknown. Brain stems of Pura KOmice were significantly enlarged and were about three times as heavy as those of the normalmice. The hippocampus and sensoric cortex of Pura KO mice were also enlarged but haddecreased weight. Moreover, more lipids were observed not only in the brain of Pura KOmice but also in blood according to blood smear analysis. It has also been shown that female Pura KO mice had more fat droplets in blood than males. Apart from the phenotypesdescribed above, Pura KO mice displayed a different weight development curve as comparedto normal mice: With the onset of tremor phenotype, Pura KO mice started to loose theirweight; the weight loss of the male KO mice beginning from postnatal day 11-15 was moreseverer than females and their weight recovery lasted longer than females; the female PuraKO mice are heavier than males while normal male mice are heavier than females, suggestingthat this obesity effect may also be influenced by animal gender. These results indicate thatPura may influence or regulate the metabolism of fat in an unknown mechanism.In the second part of this study, a new, yet uncharacterized in vitro enzymatic activity of Purawas investigated. Pura displayed a helix destabilizing activity by unwinding a labeled 17-meroligonucleotide annealed to ssM13 DNA. This activity was independent of ATP or any othernucleosides and lacks any directionality, distinguishing it therefore from common helicaseactivities. 16 truncated GST-Pura were generated by PCR for mapping of the domainresponsible for the DNA unwinding activity. It was revealed that the central part of theprotein from amino acids 54-215 was essential for this feature of Pura. The identical part ofPura was sufficient and essential for DNA binding activity. Therefore, DNA binding couldnot be separated from DNA unwinding capacity. In addition, from pull-down assays, it wasshown that the same part of the protein was also sufficient and necessary for Pura selfassociation.Since Pura has been shown to influence many important cellular processes, theDNA unwinding activity of the protein may establish a mechanism of how Pura functions indifferent cellular processes, such as replication and transcription.