Abstract
The majority of DNA polymerase alpha and primase activities bound to the nuclear matrix of regenerating rat liver were released into an extract by a mild sonication procedure. During maximal in vivo replication (22-h posthepatectomy) most of the solubilized alpha-polymerase and primase cosedimented at approximately 100 and 150 S as discrete megacomplexes with smaller amounts at 10 and 17 S. In contrast, high salt extracts obtained during nuclear matrix isolation as well as matrix extracts prepared just before the onset of in vivo replication (14-h posthepatectomy) were completely devoid of megacomplexes. In vitro incubation of the matrix extracts resulted in rapid dissolution of the megacomplexes to the 10 and 17 S forms. These relationships lead us to propose a dynamic assembly of the eucaryotic replisome which is initiated pre-replicatively as 10 and 17 S complexes and functionally expressed during in vivo replication as 100 and 150 S megacomplexes or "clustersomes."
Highlights
Richardson, 1980; Bouteille et al, 1983; Hancock, 1983; Berezney, 1984)
As a step toward identifying and characterizing these putative nuclear matrix-bound replisomes, we report the solubilization of DNA polymerase a and primase from regeneratingrat liver nuclearmatrix.Wefindthat most of the matrix-released a-polymerase and primase aroerganized into discrete 100 and 150 S megacomplexes
As a step toward themolecular elucidation of the putative replicational machinery associated with the nuclear matrix (Smith and Berezney, 1980, 1982, 1983; Jones and Su, 1982; Berezney, 1984, 1985; Nishizawa et al, 1984; Fosterand Collins, 1985; Wood and Collins, 1986) we have developed a method for solubilization of matrix-bound replicational complexes containing DNApolymerase LY and primaseactivities
Summary
Were immediately immersed in liquid Nz and stored a t -70°C until assayed. Samples from the high salt extract were loaded, centrifuged, fractionated, and stored as described above. DNA Polymerase a-Total DNA polymerase a activity was assayed (Smith and Berezney, 1982) in a 50-p1 reaction mix containing the appropriate nuclear fraction, 50 mM Tris, pH7.2,600 pg/ml DNase- This enabled very rapid preparation of the nuclear matrix and matrix extracts andavoided possibleperturbation effects of detergent treatment. While significant amounts of primase activity cosedimented with the 10 and 17S polymerase peaks, free primase activity a t about 6.5 S was the most prominent component (Fig. 4A) These results, along with previous findings that a-polymerase-primase complexes purified from the cytosol or salt extracts of whole cells generally sediment in the range of 7-10 S (Yagura et al, 1983; Chang et al, 1984; Wahl et al, 1984), suggest that the large 100 and 150 S megacomplexes are unique to thenuclear matrix fraction
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
