Nuclear matrix-bound DNA primase. Elucidation of an RNA priming system in nuclear matrix isolated from regenerating rat liver.
Recent findings in purified systems demonstrate the universality of DNA polymerase-primase complexes which may function in the priming and continuation of eucaryotic DNA replication. In this report we characterize an in vitro, nuclear matrix-associated, priming and continuation system that can utilize either endogenous matrix-bound DNA or exogenous single-stranded DNA as template. 30-40% of total nuclear DNA primase activity was recovered in association with the isolated nuclear matrix fraction from regenerating rat liver. Matrix-bound primase catalyzed the alpha-amanitin, actinomycin D-resistant synthesis of oligonucleotide chains of 8-50 nucleotides on the endogenous template. At least a portion of the RNA primers were continued by DNA polymerase alpha with deoxynucleoside triphosphate incorporation up to 300-600 nucleotides. Nearest neighbor analysis revealed ribodeoxynucleotide covalent linkages in these RNA-DNA chains. The matrix-bound primase preferred single-stranded fd DNA as exogenous template over synthetic homopolymers and was strictly dependent on the presence of ribonucleoside triphosphates. Appropriate subfractionation revealed that the matrix-bound primase activity is exclusively localized in the nuclear matrix interior. The ability of primase and DNA polymerase to synthesize covalently linked RNA-DNA products demonstrates the potentially useful role of the nuclear matrix in vitro system for elucidating the organizational and functional properties of the eucaryotic replication apparatus in the cell nucleus.
D000818:Animals; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000201:enzymology* / Q000648:ultrastructure; D019915:DNA Primase; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008115:Liver Regeneration; D012313:RNA / Q000235:genetics; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D012265:Ribonucleotides / Q000378:metabolism; D013698:Templates, Genetic
D012265:Ribonucleotides; D012313:RNA; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases