Direct Visualization of RNA-DNA Primer Removal from Okazaki Fragments Provides Support for Flap Cleavage and Exonucleolytic Pathways in Eukaryotic Cells.
During DNA replication in eukaryotic cells, short single-stranded DNA segments known as Okazaki fragments are first synthesized on the lagging strand. The Okazaki fragments originate from ∼35-nucleotide-long RNA-DNA primers. After Okazaki fragment synthesis, these primers must be removed to allow fragment joining into a continuous lagging strand. 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. One obstacle to elucidating Okazaki fragment processing has been the lack of methods that can directly examine primer removalin vivoIn this study, we developed an electron microscopy assay that can visualize nucleotide flap structures on DNA replication forks in fission yeast (Schizosaccharomyces pombe). With this assay, we first demonstrated the generation of flap structures during Okazaki fragment processingin vivoThe mean and median lengths of the flaps in wild-type cells were ∼51 and ∼41 nucleotides, respectively. We also used yeast mutants to investigate the impact of deleting key DNA replication nucleases on these flap structures. Our results provided directin vivoevidence for a previously proposed flap cleavage pathway and the critical function of Dna2 and Fen1 in cleaving these flaps. In addition, we found evidence for another previously proposed exonucleolytic pathway involving RNA-DNA primer digestion by exonucleases RNase H2 and Exo1. 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.
From the Peking-Tsinghua Center for Life Sciences, The National Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing 100871, China.
Bochao
B
Liu
From the Peking-Tsinghua Center for Life Sciences, The National Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing 100871, China.
Jiazhi
J
Hu
From the Peking-Tsinghua Center for Life Sciences, The National Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing 100871, China.
Jingna
J
Wang
From the Peking-Tsinghua Center for Life Sciences, The National Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing 100871, China kongdc@pku.edu.cn.
Daochun
D
Kong
D004247:DNA / Q000032:analysis / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D017931:DNA Primers / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004271:DNA, Fungal / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D045585:Flap Endonucleases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D012313:RNA / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000166:cytology* / Q000235:genetics / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
D017931:DNA Primers; D004271:DNA, Fungal; C022262:Okazaki fragments; C034477:RNA primers; D029702:Schizosaccharomyces pombe Proteins; D012313:RNA; D004247:DNA; C493531:Dna2 protein, S pombe; D004706:Endodeoxyribonucleases; D045585:Flap Endonucleases; C445562:Rad2 protein, S pombe
DNA enzyme; DNA replication; DNA structure; Dna2; Exo1; Fen1; Okazaki fragment processing; deoxyribonuclease (DNase); endonuclease; flap structures