5710 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Biochemistry zyxwvu 1987, 26, 57 10-57 18 Enhanced Processivity of Nuclear Matrix Bound DNA Polymerase zy a from Regenerating Rat Livert Ross A. Tubo,l Albert0 M. Martelli, and Ronald Berezney* Received April 10, 1987 Department of Biological Sciences, State University of New York at Buffalo, Buffalo. New York 14260 ABSTRACT: Translocation of DNA during in vitro DNA synthesis on nuclear matrix bound replicational assemblies from regenerating rat liver was determined by measuring the processivity (average number of nucleotides added following one productive binding event of the polymerase to the DNA template) of nuclear matrix bound D N A polymerase a with poly(dT).oligo(A) as template primer. The matrix-bound polymerase had an average processivity (28.4 nucleotides) that was severalfold higher than the bulk nuclear DNA polymerase zyxwvutsrqpo a activity extracted during nuclear matrix preparation (8.9 nucleotides). ATP at 1 mM markedly enhanced the activity and processivity of the matrix-bound polymerase but not the corresponding salt-soluble enzyme. The majority of the ATP-dependent activity and processivity enhancement was completed by 100 FM ATP and included products ranging up to full template length (1000-1200 nucleotides). Average processivity of the net ATP-stimulated polymerase activity exceeded 80 nucleotides with virtually all the DNA products >50 nucleotides. Release of nuclear matrix bound DNA polymerase a! by sonication resulted in a loss of ATP stimulation of activity and a corresponding decrease in processivity to a level similar to that of the salt-soluble polymerase (6.8 nucleotides). All nucleoside di- and triphosphates were as effective as ATP. Stimulation of both activity and processivity by the nonhydrolyzable ATP analogues adenosine zy 5’-0-(3-thiotriphosphate), 5’-adenylyl imidodiphosphate, and adenosine zyxwv 5’-0-( 1 -thiotriphosphate) further suggested that the hydrolysis of ATP is not required for enhancement to occur. A degree of specificity of nucleotide activation was indicated by the inability of nucleoside monophosphates, the ATP analogues P1 ,P-bis(S’-adenosyl) tetraphosphate and 5’-adenylyl methylenediphosphate, the ADP-nonhydrolyzable analogue adenosine 5’-0-(2-thiodiphosphate), and pyrophosphate to enhance either activity or processivity. S e v e r a l critical biological processes occurring within the eucaryotic cell nucleus have been shown to be closely associated with the nuclear matrix (Berezney, 1984). This dynamic proteinaceous nuclear framework is obtained following nu- clease, salt, and detergent treatments of isolated nuclei (Berezney, 1984). Chromatin within the cell nucleus is or- ganized into a series of supercoiled loops (Cook zyxwvuts & Brezell, 1975; Benyajati & Worcel, 1976; Georgiev et al., 1978; Igo- Kemenes & Zachau, 1978), which are attached to the nuclear matrix of interphase nuclei (Wanka et al., 1977; Vogelstein et al., 1980; Berezney & Buchholtz, 1981a). A number of in vivo incorporation studies suggest that the sites of eucaryotic chromosomal replication are closely associated with the isolated nuclear matrix structure (Berezney & Coffey, 1975, 1976; Dijkwel et al., 1979, 1986; McCready et al., 1980; Pardoll et al., 1980; Berezney & Buchholtz, 1981b; Aelen et al., 1983; van der Valden et al., 1984; Carri et al., 1986; Razin et al., 1986). It is proposed that during eucaryotic DNA replication DNA loops and nuclear matrix bound replicational complexes or replisomes translocate relative to each other (Dijkwel et al., 1979; McCready et al., 1980; Pardoll et al., 1980; Berezney & Buchholtz, 1981b). Consistent with this nuclear matrix replisome model, DNA polymerase a, DNA primase, and other replicative enzymes are associated with the nuclear matrix in a cell cycle dependent fashion (Smith & Berezney, 1980, 1982, 1983; Foster & Collins, 1985; Wood & Collins, 1986; Tubo & Berezney, 1987a), and at least a major portion of the in vitro DNA synthesis on endogenous matrix-attached ‘This work was supported by National Institutes of Health Grant *Address correspondence to this author. GM-23922 awarded to R.B. Present address: Dana Farber Cancer Institute, Harvard Medical School, Boston, MA 021 15. 0006-2960/87/0426-57 10$01.50/0 DNA represents a continuation at in vivo initiated replicational forks (Tubo et al., 1985). Moreover, the nuclear matrix bound DNA polymerase a cosediments on sucrose gradients with several other replicative enzymes following solubilization from the matrix structure (Tubo & Berezney, 1987a). While the relationship of these matrix-solubilized multienzyme replica- tional complexes to functional sites of DNA replication in the cell nucleus remains to be clarified, it is interesting to note that recent immunocytochemical findings indicate that both DNA polymerase a and the sites of active in vivo replication are arranged in discrete granular clusters within the cell nu- cleus (Yamamoto et al., 1984; Nakamura et al., 1984, 1986). Moreover, the granular clusters or putative “replication centers” observed in intact cells were maintained following extractions used for obtaining nuclear matrix structures (Yamamoto et al., 1984). In this study we have evaluated the ability of the nuclear matrix bound replisomes to translocate DNA by measuring the processivity of DNA polymerase a. Processivity is defined as the number of nucleotides added to a DNA template during a single binding-translocation-dissociation cycle of a polym- erase (Bambara et al., 1978; Fay et al., 1981; Hockensmith & Bambara, 1981). We demonstrate that the matrix-bound enzyme is severalfold more processive in DNA synthesis then the corresponding salt-soluble polymerase of the cell nucleus. In addition, ATP and other nucleoside di- and triphosphates stimulate enzyme activity 2-3-fold and markedly enhance the processivity to DNA products up to full template length (1000-1200 nucleotides). Release of the polymerase from the nuclear matrix structure results in a loss in ATP stimulation of activity and a large decrease in processivity to a level similar to the bulk salt-soluble polymerase activity and most purified preparations of DNA polymerase (Y (Das & Fujimura, 1979; Fisher et al., 1979; Detera et al., 1981; Hockensmith & zyxwvutsrqp 0 1987 American Chemical Society