Characterization of RNA primers synthesized by the human breast cancer cell DNA synthesome.
We previously reported on the purification and characterization of a functional multi-protein DNA replication complex (the DNA synthesome) from human cells and tissues. The synthesome is fully competent to carry-out all phases of the DNA replication process in vitro. In this study, DNA primase, a component of the synthesome, is examined to determine its activity and processivity in the in vitro synthesis and extension of RNA primers. Our results show that primase activity in the P4 fraction of the synthesome is 30-fold higher than that of crude cell extracts. The synthesome synthesizes RNA primers that are 7-10 ribonucleotides long and DNA primers that are 20-40 deoxyribonucleotides long using a poly(dT) template of exogenous single-stranded DNA. The synthesome-catalyzed RNA primers can be elongated by E. coli DNA polymerase I to form the complementary DNA strands on the poly(dT) template. In addition, the synthesome also supports the synthesis of native RNA primers in vitro using an endogenous supercoiled double-stranded DNA template. Gel analysis demonstrates that native RNA primers are oligoribonucleotides of 10-20 nt in length and the primers are covalently link to DNA to form RNA-primed nascent DNA of 100-200 nt. Our study reveals that the synthesome model is capable of priming and continuing DNA replication. The ability of the synthesome to synthesize and extend RNA primers in vitro elucidates the organizational and functional properties of the synthesome as a potentially useful replication apparatus to study the function of primase and the interaction of primase with other replication proteins.
Dai
Heqiao
H
Department of Medicine, Division of Hematology/Oncology, Indiana University School of Medicine, Indiana University Cancer Research Institute, Indiana University Simon Cancer Center, 1044 W. Walnut Street R4-170 Indianapolis, Indiana 46202, USA.
Liu
Jianying
J
Malkas
Linda H
LH
Hickey
Robert J
RJ
D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D019915:DNA Primase / Q000378:metabolism; D017931:DNA Primers; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005260:Female; D006801:Humans; D009097:Multienzyme Complexes / Q000378:metabolism*; D012313:RNA / Q000096:biosynthesis*; D000099272:Replisomes
D019915:DNA Primase; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase; D009097:Multienzyme Complexes; D012313:RNA; D000099272:Replisomes; C034477:RNA primers