title: Creation and removal of embedded ribonucleotides in chromosomal DNA during mammalian Okazaki fragment processing.
abstract: Mammalian RNase HI has been shown to specifically cleave the initiator RNA of Okazaki fragments at the RNA-DNA junction, leaving a single ribonucleotide attached to the 5'-end of the downstream DNA segment. This monoribonucleotide can then be removed by the mammalian 5'- to 3'-exo-/endonuclease, a RAD2 homolog-1 (RTH-1) class nuclease, also known as flap endonuclease-1 (FEN-1). Although FEN-1/RTH-1 nuclease often requires an upstream primer for efficient activity, the presence of an upstream primer is usually inhibitory or neutral for removal of this 5'-monoribonucleotide. Using model Okazaki fragment substrates, we found that DNA ligase I can seal a 5'-monoribonucleotide into DNA. When both ligase and FEN-1/RTH-1 were present simultaneously, some of the 5'-monoribonucleotides were ligated into DNA, while others were released. Thus, a 5'-monoribonucleotide, particularly one that is made resistant to FEN-1/RTH-1-directed cleavage by extension of an inhibitory upstream primer, can be ligated into the chromosome, despite the presence of FEN-1/RTH-1 nuclease. DNA ligase I was able to seal different monoribonucleotides into the DNA for all substrates tested, with an efficiency of 1-13% that of ligating DNA. These embedded monoribonucleotides can be removed by the combined action of RNase HI, cutting on the 5'-side, and FEN-1/RTH-1 nuclease, cleaving on the 3'-side. After FEN-1/RTH-1 action and extension by polymerization, DNA ligase I can join the entirely DNA strands to complete repair.
authors.affiliation: Department of Biochemistry, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
authors.forename: J A
authors.initials: JA
authors.lastname: Rumbaugh
authors.forename: R S
authors.initials: RS
authors.lastname: Murante
authors.forename: S
authors.initials: S
authors.lastname: Shi
authors.forename: R A
authors.initials: RA
authors.lastname: Bambara
mesh_terms: D000818:Animals; D002417:Cattle; D002875:Chromosomes; D004247:DNA / Q000378:metabolism*; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism; D017931:DNA Primers; D004260:DNA Repair; D004261:DNA Replication; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D016914:Ribonuclease H / Q000378:metabolism; D012265:Ribonucleotides / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; C022262:Okazaki fragments; D012265:Ribonucleotides; D004247:DNA; D016914:Ribonuclease H; D011088:DNA Ligases; D000072481:DNA Ligase ATP