Discontinuous DNA synthesis by purified mammalian proteins.
Five proteins purified from mouse cells acting together efficiently convert a single-stranded circular DNA template to covalently closed duplex circle by a discontinuous mechanism. DNA polymerase alpha/primase with the assistance of alpha accessory factor covers the single-stranded circle with RNA-primed DNA fragments. Primers are removed by a combination of RNase H-1 and a 5'-exonuclease that was identified by its ability to complete this in vitro system. The 5'-exonuclease is required to remove residual one or two ribonucleotides at the primer/DNA junction that are resistant to RNase H-1. Gap filling is by the DNA polymerase alpha/primase, and DNA ligase I converts the DNA fragments to continuous strand. The concerted action of the five proteins emulates synthesis of the staging strand at the replication fork.
Goulian
M
M
Department of Medicine, University of California, San Diego, La Jolla 92093-0613.
Richards
S H
SH
Heard
C J
CJ
Bigsby
B M
BM
D000818:Animals; D004247:DNA / Q000096:biosynthesis*; D011088:DNA Ligases / Q000378:metabolism; D004257:DNA Polymerase II / Q000378:metabolism*; D019915:DNA Primase; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004722:Endoribonucleases / Q000378:metabolism; D005092:Exonucleases / Q000302:isolation & purification / Q000378:metabolism; D066298:In Vitro Techniques; D007939:Leukemia L1210; D051379:Mice; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012316:RNA Nucleotidyltransferases / Q000378:metabolism; D016914:Ribonuclease H; D013698:Templates, Genetic; D014407:Tumor Cells, Cultured
D004277:DNA, Single-Stranded; D009687:Nuclear Proteins; D004247:DNA; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004257:DNA Polymerase II; D004722:Endoribonucleases; D005092:Exonucleases; D016914:Ribonuclease H; D011088:DNA Ligases