Direct Visualization of RNA-DNA Primer Removal from Okazaki Fragments   Provides Support for Flap Cleavage and Exonucleolytic Pathways in   Eukaryotic Cells

doi:10.1074/jbc.M116.758599

CORC > 北京大学 > 生命科学学院

	Direct Visualization of RNA-DNA Primer Removal from Okazaki Fragments Provides Support for Flap Cleavage and Exonucleolytic Pathways in Eukaryotic Cells
	Liu, Bochao ; Hu, Jiazhi ; Wang, Jingna ; Kong, Daochun
刊名	JOURNAL OF BIOLOGICAL CHEMISTRY
	2017
关键词	NUCLEOTIDE EXCISION-REPAIR STALLED REPLICATION FORKS SACCHAROMYCES-CEREVISIAE IN-VITRO POLYMERASE-EPSILON NUCLEAR ANTIGEN YEAST ENDONUCLEASE-1 MATURATION PROTEIN
DOI	10.1074/jbc.M116.758599
英文摘要	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 similar to 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 RNADNA 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 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). 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 similar to 51 and similar to 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 direct in vivo evidence 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 H2and Exo1. Taken together, our observations suggest a dual mechanism for Okazaki fragment maturation in lagging strand synthesis and establish; National Natural Science Foundation of China [31230021, 31400674]; Ministry of Science and Technology of China [2013CB911000]; Peking-Tsinghua Center for Life Sciences; School of Life Sciences; National Key Laboratory of Protein and Plant Gene Research; SCI(E); ARTICLE; 12; 4777-4788; 292
语种	英语
内容类型	期刊论文
源URL	[http://ir.pku.edu.cn/handle/20.500.11897/474494]
专题	生命科学学院
推荐引用方式 GB/T 7714	Liu, Bochao,Hu, Jiazhi,Wang, Jingna,et al. Direct Visualization of RNA-DNA Primer Removal from Okazaki Fragments Provides Support for Flap Cleavage and Exonucleolytic Pathways in Eukaryotic Cells[J]. JOURNAL OF BIOLOGICAL CHEMISTRY,2017.
APA	Liu, Bochao,Hu, Jiazhi,Wang, Jingna,&Kong, Daochun.(2017).Direct Visualization of RNA-DNA Primer Removal from Okazaki Fragments Provides Support for Flap Cleavage and Exonucleolytic Pathways in Eukaryotic Cells.JOURNAL OF BIOLOGICAL CHEMISTRY.
MLA	Liu, Bochao,et al."Direct Visualization of RNA-DNA Primer Removal from Okazaki Fragments Provides Support for Flap Cleavage and Exonucleolytic Pathways in Eukaryotic Cells".JOURNAL OF BIOLOGICAL CHEMISTRY (2017).