Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Mar 24;292(12):4777-4788.
doi: 10.1074/jbc.M116.758599. Epub 2017 Feb 3.

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

Bochao Liu  1 , Jiazhi Hu  1 , Jingna Wang  1 , Daochun Kong  2
Affiliations

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

Bochao Liu et al. J Biol Chem. .

Abstract

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 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 ∼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 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 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.

Keywords: DNA enzyme; DNA replication; DNA structure; Dna2; Exo1; Fen1; Okazaki fragment processing; deoxyribonuclease (DNase); endonuclease; flap structures.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

FIGURE 1.
FIGURE 1.
Flap structures in WT, fen1−, dna2−, and fen1−-dna2− cells. A, a schematic of the flap and exonuclease pathways. 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. The flap structures are subsequently cleaved by flap endonucleases, such as Dna2 and Fen1, to generate ligatable nicks. In the exonuclease pathway, the RNA-DNA primers are directly digested by RNase H2 and an exonuclease to generate ligatable nicks. Finally, DNA ligase I joins these processed Okazaki fragments together, and an intact lagging strand is produced. B–P, EM images of replication forks from WT, fen1−, dna2−, and fen1−-dna2− cells. The cells were not synchronized, and the replication forks for EM imaging were not enriched by BND-cellulose. WT and fen1− cells were grown at 30 °C. dna2− and fen1−-dna2− cells were obtained from germinated dna2− and fen1−-dna2− spores at 30 °C. B–D, replication forks from WT cells. E–J, replication forks from fen1− cells. I–L, replication forks from dna2− cells. M–P, replication forks from fen1−-dna2− cells. The flap structures are indicated by black arrows. H, the black triangles indicate unwound strands. N, the empty triangle indicates the regressed arm (reversed forks).
FIGURE 2.
FIGURE 2.
Statistics of flap structures in replication forks. A, the percentage of forks harboring flaps. B, the average distance between flaps in the WT, fen1−, dna2−, and fen1−-dna2− replication forks. C, the mean and median lengths of the flaps in WT, fen1−, dna2−, and fen1−-dna2− replication forks. The distribution of flap lengths is shown in the left panel. The significance of the differences in the flap lengths among WT and mutant cells is shown in the table of statistical significance. D, the distribution of flaps on one or two strands of the forks. E, the percentages of the flap distribution on DNA strands from the fork end. Error bars indicate the standard error.
FIGURE 3.
FIGURE 3.
RPA foci in WT, fen1−, and dna2ts cells. A, a schematic of cells in M-G1, S, early G2, and late G2 phases. The cell phases were determined based on the cell length and number of nuclei present in an S. pombe cell. B, RPA foci (10×100-fold amplification) measurements in WT, fen1−, and dna2ts cells. SSB1, the largest subunit of RPA, was tagged by YFP. The ssb1-yfp gene was integrated into the ssb1 gene locus of the WT, fen1−, and dna2ts strains. WT, fen1−, and dna2ts cells expressing SSB1-YFP were cultured to log phase at 30 °C, and RPA foci were examined. C–F, RPA foci were examined during M-G1, S, early G2, and late G2 phases in WT, fen1−, and dna2ts cells. Cell growth conditions and the number of cells examined are indicated. For each statistic, at least four independent experiments were conducted. Error bars indicate the standard error. DIC, differential interference contrast.
FIGURE 4.
FIGURE 4.
Flap structures in the rnh201−, exo1−, exo1−-rnh201−, and fen1−-rnh201− cells. A–I, EM images of replication forks from rnh201−, exo1−, exo1−-rnh201−, and fen1−-rnh201− cells. The experiments were conducted as in Fig. 1: the cells were not synchronized, and the replication forks for EM imaging were not enriched by BND-cellulose. All cells were grown at 30 °C. A and B, replication forks from rnh201− cells. C and D, replication forks from exo1− cells. E and F, replication forks from exo1−-rnh201− cells. G–I, replication forks from fen1−-rnh201− cells. The flap structures are indicated by black arrows.
FIGURE 5.
FIGURE 5.
Statistics of flap structures in the rnh201−, exo1−, exo1−-rnh201−, and fen1−-rnh201− replication forks. A, the percentage of forks harboring flaps. B, the average distance between flaps in the rnh201−, exo1−, exo1−-rnh201−, and fen1−-rnh201− replication forks. C, the mean and median lengths of the flaps in rnh201−, exo1−, exo1−-rnh201−, and fen1−-rnh201− replication forks. The distribution of flap lengths is shown in the left panel. The significance of the differences in the flap lengths among WT and mutant cells is shown in the table of statistical significance. D, the distribution of flaps on one or two strands of the forks. E, the percentages of the flap distribution on DNA strands from the fork end. Error bars indicate the standard error.
FIGURE 6.
FIGURE 6.
Diagrams demonstrating the processing of Okazaki fragments. The flap pathway and the exonuclease pathway for the removal of RNA-DNA primers from Okazaki fragments are shown. i, the RNase H2- and Exo1-mediated exonuclease pathway. The RNA-DNA primers are directly hydrolyzed by RNase H2 and Exo1. DNA pol δ catalyzes DNA synthesis to fill in the remaining DNA gaps generated by RNA-DNA primer removal. Finally, DNA ligase I (Lig I) seals the nicks, and an intact lagging strand is generated. ii, the long flap pathway. The RNA-DNA primers are first displaced by pol δ-mediated displacement DNA synthesis and subsequently generate long flap structures that are coated by RPA. Dna2 first comes to cleave the long flaps, which leaves a flap of 5–7 nt. Then Fen1 cleaves the remaining short flaps to generate ligatable nicks. iii, the short flap pathway. The pol δ-mediated displacement DNA synthesis creates a short flap, and this short flap is cleaved by Fen1. Through a few cycles, the RNA-DNA primer is completely removed.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Pursell Z. F., Isoz I., Lundström E. B., Johansson E., and Kunkel T. A. (2007) Yeast DNA polymerase ϵ participates in leading-strand DNA replication. Science 317, 127–130 - PMC - PubMed
    1. Nick McElhinny S. A., Gordenin D. A., Stith C. M., Burgers P. M., and Kunkel T. A. (2008) Division of labor at the eukaryotic replication fork. Mol. Cell 30, 137–144 - PMC - PubMed
    1. Sakabe K., and Okazaki R. (1966) A unique property of the replicating region of chromosomal DNA. Biochim. Biophys. Acta 129, 651–654 - PubMed
    1. Smith D. J., and Whitehouse I. (2012) Intrinsic coupling of lagging-strand synthesis to chromatin assembly. Nature 483, 434–438 - PMC - PubMed
    1. Bullock P. A., Seo Y. S., and Hurwitz J. (1991) Initiation of simian virus 40 DNA synthesis in vitro. Mol. Cell. Biol. 11, 2350–2361 - PMC - PubMed

MeSH terms