title: Enzymatic completion of mammalian lagging-strand DNA replication.
abstract: Using purified proteins from calf and a synthetic substrate, we have reconstituted the enzymatic reactions required for mammalian Okazaki fragment processing in vitro. The required reactions are removal of initiator RNA, synthesis from an upstream fragment to generate a nick, and then ligation. With our substrate, RNase H type I (RNase HI) makes a single cut in the initiator RNA, one nucleotide 5' of the RNA-DNA junction. The double strand specific 5' to 3' exonuclease removes the remaining monoribonucleotide. After dissociation of cleaved RNA, synthesis by DNA polymerase generates a nick, which is then sealed by DNA ligase I. The unique specificities of the two nucleases for primers with initiator RNA strongly suggest that they perform the same reactions in vivo.
authors.affiliation: Department of Biochemistry and Molecular Biology, Wright State University, Dayton, OH 45435.
authors.forename: J J
authors.initials: JJ
authors.lastname: Turchi
authors.forename: L
authors.initials: L
authors.lastname: Huang
authors.forename: R S
authors.initials: RS
authors.lastname: Murante
authors.forename: Y
authors.initials: Y
authors.lastname: Kim
authors.forename: R A
authors.initials: RA
authors.lastname: Bambara
mesh_terms: D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism*; D017931:DNA Primers; D004261:DNA Replication; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D008322:Mammals; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D012313:RNA / Q000378:metabolism; D016914:Ribonuclease H / Q000302:isolation & purification / Q000378:metabolism*; D013950:Thymus Gland / Q000201:enzymology
chemical_list: D017931:DNA Primers; D012313:RNA; D004247:DNA; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; D016914:Ribonuclease H; D011088:DNA Ligases; D000072481:DNA Ligase ATP