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

Abstract

Edited by Patrick SungDuring DNA replication in eukaryotic cells, short singlestranded DNA segments known as Okazaki fragments are first synthesized on the lagging strand. The Okazaki fragments originate from ϳ35-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 ϳ51 and ϳ41 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.In eukaryotic cells, DNA pol 2 ⑀ and pol ␦ direct the synthesis of leading and lagging strand DNA, respectively (1, 2). The antiparallel nature of DNA and the unique 5Ј to 3Ј direction of DNA synthesis by all DNA polymerases make the synthesis of leading strand continuous and lagging strand discontinuous. In the lagging strand, Okazaki fragments are synthesized first (3). The average size of Okazaki fragments in eukaryotic cells is ϳ150 -200 nucleotides (nt) (4). Because DNA polymerases lack de novo DNA synthesis activity, each Okazaki fragment contains an RNA-DNA primer at its 5Ј-end, and this primer is synthesized with low fidelity by primase-DNA pol ␣ complex (5-7). DNA ligase I is responsible for joining Okazaki fragments together to form a continuous lagging strand. Because DNA ligase I is unable to join DNA to RNA, the RNA-DNA primers must be removed from each Okazaki fragment to complete lagging strand DNA synthesis and maintain genomic stability.The mechanism underlying the removal of RNA-DNA primers from Okazaki fragments remains uncertain. Over the previous 20 years, three models have been proposed to explain how these primers are removed (8). In the first model, the RNA-DNA primers are hydrolyzed directly by RNase H2 and DNA exonucleases, such as Fen1 (the exonu...

Phosphate steering by Flap Endonuclease 1 promotes 5′-flap specificity and incision to prevent genome instability

“…In the wt strain, median expansion size corresponded to 47 triplets49. The rad27 knockout was different: median expansion size was 32 repeats, and Kolmogorov-Smirnov (KS) comparison confirms a significant difference from the wt strain ( P <0.001), which agrees with known flap size in FEN1 knockouts54. The expansion scale in near-catalytic-dead (D179A) and 4E Rad27 mutants lies between the wt and knockout mutant: the median is 40 repeats and KS shows significant difference from wt ( P <0.05).…”

On the wrong DNA track: Molecular mechanisms of repeat-mediated genome instability

“…However, the same mutations also increase repeat contraction rates (183, 289, 307, 309, 310, 312-314, 316 -318), a phenomenon that cannot be explained by the flap incorporation model. A more general model suggests that in the absence of fully functional Rad27 or Dna2, the genomewide accumulation of long ssDNA species (314,320,321), such as flaps or gaps, titrates the ssDNA-binding protein replication protein A (RPA) from the repetitive regions (322). Due to the lack of RPA, transiently formed ssDNA within the repetitive tracts is likely to equilibrate into DNA secondary structures, which trigger repeat instability ( Fig.…”

Processing of eukaryotic Okazaki fragments by redundant nucleases can be uncoupled from ongoing DNA replicationin vivo

“…Dna2 or Rad27) is required for cells to progress 234 through S-phase. We reasoned that the requirement for endonucleolytic Okazaki fragment flap 235 processing during S-phase could arise from the accumulation of a small number of long, 236 unprocessed flaps that might persist in CLB2-RAD27 CLB2-DNA2 cells (Liu et al, 2017). These 237 long flaps could deplete cellular RPA pools, leading to checkpoint-mediated cell cycle arrest 238 (Byun et al, 2005) and/or catastrophic failure of replication (Toledo et al, 2013).…”