![]() Finally, DNA ligase I joins these processed Okazaki fragments together, and an intact lagging strand is produced. In the exonuclease pathway, the RNA-DNA primers are directly digested by RNase H2 and an exonuclease to generate ligatable nicks. The flap structures are subsequently cleaved by flap endonucleases, such as Dna2 and Fen1, to generate ligatable nicks. As suggested by the flap pathway, the low fidelity DNA pol α-primase-synthesized RNA-DNA primers are displaced by DNA pol δ-mediated displacement DNA synthesis and subsequently generate flap structures. A, a schematic of the flap and exonuclease pathways. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.įlap structures in WT, fen1 −, dna2 −, and fen1 −- dna2 − cells. Taken together, our observations suggest a dual mechanism for Okazaki fragment maturation in lagging strand synthesis and establish a new strategy for interrogation of this fascinating process.ĭNA enzyme DNA replication DNA structure Dna2 Exo1 Fen1 Okazaki fragment processing deoxyribonuclease (DNase) endonuclease flap structures. In addition, we found evidence for another previously proposed exonucleolytic pathway involving RNA-DNA primer digestion by exonucleases RNase H2 and Exo1. Our results provided direct in vivo evidence for a previously proposed flap cleavage pathway and the critical function of Dna2 and Fen1 in cleaving these flaps. We also used yeast mutants to investigate the impact of deleting key DNA replication nucleases on these flap structures. With this assay, we first demonstrated the generation of flap structures during Okazaki fragment processing in vivo The mean and median lengths of the flaps in wild-type cells were ∼51 and ∼41 nucleotides, respectively. One obstacle to elucidating Okazaki fragment processing has been the lack of methods that can directly examine primer removal in vivo In this study, we developed an electron microscopy assay that can visualize nucleotide flap structures on DNA replication forks in fission yeast ( Schizosaccharomyces pombe). ![]() To date, the models of enzymatic machinery that removes the RNA-DNA primers have come almost exclusively from biochemical reconstitution studies and some genetic interaction assays, and there is little direct evidence to confirm these models. After Okazaki fragment synthesis, these primers must be removed to allow fragment joining into a continuous lagging strand. ![]() The Okazaki fragments originate from ∼35-nucleotide-long RNA-DNA primers. ![]() During DNA replication in eukaryotic cells, short single-stranded DNA segments known as Okazaki fragments are first synthesized on the lagging strand. ![]()
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