Enhanced processivity of nuclear matrix bound DNA polymerase alpha from regenerating rat liver.
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 DNA polymerase alpha with poly(dT).oligo(A)10 as template primer. The matrix-bound polymerase had an average processivity (28.4 nucleotides) that was severalfold higher than the bulk nuclear DNA polymerase alpha 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 microM 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 greater than 50 nucleotides. Release of nuclear matrix bound DNA polymerase alpha 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 5'-O-(3-thiotriphosphate), 5'-adenylyl imidodiphosphate, and adenosine 5'-O-(1-thiotriphosphate) further suggested that the hydrolysis of ATP is not required for enhancement to occur.(ABSTRACT TRUNCATED AT 250 WORDS)
Tubo
R A
RA
Department of Biological Sciences, State University of New York at Buffalo, Buffalo 14260.
Martelli
A M
AM
Berezney
R
R
D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology*; D004247:DNA / Q000096:biosynthesis / Q000302:isolation & purification; D004257:DNA Polymerase II / Q000302:isolation & purification / Q000378:metabolism*; D004261:DNA Replication; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008115:Liver Regeneration; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D013698:Templates, Genetic
D004247:DNA; D004257:DNA Polymerase II