Pol δ synthetizes 100–500 nucleotides long Okazaki fragments before it displaces the 5′-end of the preceding primer. Lagging strand synthesis occurs in a discontinuous manner due to the movement of polymerase in the opposite direction of the replication fork and therefore requires the use of multiple RNA/DNA primers. DNA synthesis on the leading strand requires only one primer and has mostly uninterrupted DNA polymerase activity. Strand uncoupling induced by POLA1 inhibition during DNA replication generates single-stranded DNA (ssDNA) that is tolerated by cells if it is protected by the surplus of RPA molecules. DNA synthesis at leading and lagging strands works independently and can be uncoupled in vivo ( Ercilla et al, 2020). Hybrid RNA/DNA primers then become substrates for elongating polymerases ε and δ on leading and lagging strands, respectively. DNA replication is initiated on both strands by the synthesis of hybrid RNA/DNA primer by the polymerase α complex ( Burgers, 2009), which consists of the catalytic subunit POLA1, accessory subunit POLA2, and primase subunits PRIM1 and PRIM2. In recent molecular biology research, EME was used, based on an earlier report by Burhans and colleagues ( Burhans et al, 1991), as a specific inhibitor of lagging strand synthesis which prevents the formation of Okazaki fragments (OFs), uncoupling leading and lagging strand replication ( Hanzlikova et al, 2018 Thakar et al, 2020 Xiao et al, 2020 Cong et al, 2021 Yamashita et al, 2022).ĭNA replication occurs in opposite directions on each of the two parental strands. The inhibition of protein, DNA, and RNA synthesis is EME’s main mechanism of action in biological systems ( Grollman, 1968 Gupta & Siminovitch, 1976). However, its medical use is limited mainly due to both myopathy and cardiotoxicity which are associated with chronic usage of EME ( Wang & Yang, 2020). Collectively, our study reveals that emetine is not a specific lagging strand synthesis inhibitor with implications for its use in molecular biology.Įmetine (EME), an alkaloid present in the plant Carapichea ipecacuanha, is the main active ingredient in ipecac syrup which has been used in traditional medicine as an emetic, expectorant and antiparasitic drug. Emetine and mild POLA1 inhibition prevent S-phase poly(ADP-ribosyl)ation. Thus, our results are consistent with the hypothesis that emetine affects DNA replication by proteosynthesis inhibition. Mechanistically, we demonstrate that the acute block of proteosynthesis by emetine temporally precedes its effects on DNA replication. Emetine is also an inhibitor of proteosynthesis and ongoing proteosynthesis is essential for the accurate replication of DNA. In line with this, emetine does not activate the replication checkpoint nor DNA damage response. Here, by analysis of lagging strand synthesis inhibitors, we show that despite emetine completely inhibiting DNA replication: it does not induce the generation of single-stranded DNA and chromatin-bound RPA32 (CB-RPA32). Natural alkaloid emetine is used as a specific inhibitor of lagging strand synthesis, uncoupling leading and lagging strand replication. Haruki H, Nishikawa J, Laemmli UK (2008) The anchor-away technique: rapid, conditional establishment of yeast mutant phenotypes.DNA synthesis of the leading and lagging strands works independently and cells tolerate single-stranded DNA generated during strand uncoupling if it is protected by RPA molecules. Kubota T, Nishimura K, Kanemaki MT et al (2013) The Elg1 replication factor C-like complex functions in PCNA Unloading during DNA replication. Sanchez-Diaz A, Kanemaki M, Marchesi V et al (2004) Rapid depletion of budding yeast proteins by fusion to a heat-inducible degron. Rohland N, Reich D (2012) Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture. McGuffee SR, Smith DJ, Whitehouse I (2013) Quantitative, genome-wide analysis of eukaryotic replication initiation and termination. Smith DJ, Whitehouse I (2012) Intrinsic coupling of lagging-strand synthesis to chromatin assembly. Nasmyth KA (1977) Temperature-sensitive lethal mutants in the structural gene for DNA ligase in the yeast Schizosaccharomyces pombe. Johnston LH, Nasmyth KA (1978) Saccharomyces cerevisiae cell cycle mutant cdc9 is defective in DNA ligase. Distribution of functions between FEN1 AND DNA2. Nature 369:207–212Īyyagari R, Gomes XV, Gordenin DA et al (2003) Okazaki fragment maturation in yeast. Waga S, Stillman B (1994) Anatomy of a DNA replication fork revealed by reconstitution of SV40 DNA replication in vitro. Nethanel T, Kaufmann G (1990) Two DNA polymerases may be required for synthesis of the lagging DNA strand of simian virus 40.
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