Journal articles: 'Okazaki fragments'

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Padel, Ruth. "The Okazaki Fragments." Poem 1, no. 1 (2013): 114–23. http://dx.doi.org/10.1080/20519842.2013.11415334.

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Kumamoto, Soichiro, Atsuya Nishiyama, Yoshie Chiba, et al. "HPF1-dependent PARP activation promotes LIG3-XRCC1-mediated backup pathway of Okazaki fragment ligation." Nucleic Acids Research 49, no. 9 (2021): 5003–16. http://dx.doi.org/10.1093/nar/gkab269.

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Abstract DNA ligase 1 (LIG1) is known as the major DNA ligase responsible for Okazaki fragment joining. Recent studies have implicated LIG3 complexed with XRCC1 as an alternative player in Okazaki fragment joining in cases where LIG1 is not functional, although the underlying mechanisms are largely unknown. Here, using a cell-free system derived from Xenopus egg extracts, we demonstrated the essential role of PARP1-HPF1 in LIG3-dependent Okazaki fragment joining. We found that Okazaki fragments were eventually ligated even in the absence of LIG1, employing in its place LIG3-XRCC1, which was recruited onto chromatin. Concomitantly, LIG1 deficiency induces ADP-ribosylation of histone H3 in a PARP1-HPF1-dependent manner. The depletion of PARP1 or HPF1 resulted in a failure to recruit LIG3 onto chromatin and a subsequent failure in Okazaki fragment joining in LIG1-depleted extracts. Importantly, Okazaki fragments were not ligated at all when LIG1 and XRCC1 were co-depleted. Our results suggest that a unique form of ADP-ribosylation signaling promotes the recruitment of LIG3 on chromatin and its mediation of Okazaki fragment joining as a backup system for LIG1 perturbation.

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Spiering, Michelle M., Philip Hanoian, Swathi Gannavaram, and Stephen J. Benkovic. "RNA primer–primase complexes serve as the signal for polymerase recycling and Okazaki fragment initiation in T4 phage DNA replication." Proceedings of the National Academy of Sciences 114, no. 22 (2017): 5635–40. http://dx.doi.org/10.1073/pnas.1620459114.

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The opposite strand polarity of duplex DNA necessitates that the leading strand is replicated continuously whereas the lagging strand is replicated in discrete segments known as Okazaki fragments. The lagging-strand polymerase sometimes recycles to begin the synthesis of a new Okazaki fragment before finishing the previous fragment, creating a gap between the Okazaki fragments. The mechanism and signal that initiate this behavior—that is, the signaling mechanism—have not been definitively identified. We examined the role of RNA primer–primase complexes left on the lagging ssDNA from primer synthesis in initiating early lagging-strand polymerase recycling. We show for the T4 bacteriophage DNA replication system that primer–primase complexes have a residence time similar to the timescale of Okazaki fragment synthesis and the ability to block a holoenzyme synthesizing DNA and stimulate the dissociation of the holoenzyme to trigger polymerase recycling. The collision with primer–primase complexes triggering the early termination of Okazaki fragment synthesis has distinct advantages over those previously proposed because this signal requires no transmission to the lagging-strand polymerase through protein or DNA interactions, the mechanism for rapid dissociation of the holoenzyme is always collision, and no unique characteristics need to be assigned to either identical polymerase in the replisome. We have modeled repeated cycles of Okazaki fragment initiation using a collision with a completed Okazaki fragment or primer–primase complexes as the recycling mechanism. The results reproduce experimental data, providing insights into events related to Okazaki fragment initiation and the overall functioning of DNA replisomes.

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Chen, Danqi, Hongjun Yue, Michelle M. Spiering, and Stephen J. Benkovic. "Insights into Okazaki Fragment Synthesis by the T4 Replisome." Journal of Biological Chemistry 288, no. 29 (2013): 20807–16. http://dx.doi.org/10.1074/jbc.m113.485961.

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In this study, we employed a circular replication substrate with a low priming site frequency (1 site/1.1 kb) to quantitatively examine the size distribution and formation pattern of Okazaki fragments. Replication reactions by the T4 replisome on this substrate yielded a patterned series of Okazaki fragments whose size distribution shifted through collision and signaling mechanisms as the gp44/62 clamp loader levels changed but was insensitive to changes in the gp43 polymerase concentration, as expected for a processive, recycled lagging-strand polymerase. In addition, we showed that only one gp45 clamp is continuously associated with the replisome and that no additional clamps accumulate on the DNA, providing further evidence that the clamp departs, whereas the polymerase is recycled upon completion of an Okazaki fragment synthesis cycle. We found no support for the participation of a third polymerase in Okazaki fragment synthesis.

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Cronan, Glen E., Elena A. Kouzminova, and Andrei Kuzminov. "Near-continuously synthesized leading strands inEscherichia coliare broken by ribonucleotide excision." Proceedings of the National Academy of Sciences 116, no. 4 (2019): 1251–60. http://dx.doi.org/10.1073/pnas.1814512116.

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In vitro, purified replisomes drive model replication forks to synthesize continuous leading strands, even without ligase, supporting the semidiscontinuous model of DNA replication. However, nascent replication intermediates isolated from ligase-deficientEscherichia colicomprise only short (on average 1.2-kb) Okazaki fragments. It was long suspected that cells replicate their chromosomal DNA by the semidiscontinuous mode observed in vitro but that, in vivo, the nascent leading strand was artifactually fragmented postsynthesis by excision repair. Here, using high-resolution separation of pulse-labeled replication intermediates coupled with strand-specific hybridization, we show that excision-proficientE. coligenerates leading-strand intermediates >10-fold longer than lagging-strand Okazaki fragments. Inactivation of DNA-repair activities, including ribonucleotide excision, further increased nascent leading-strand size to ∼80 kb, while lagging-strand Okazaki fragments remained unaffected. We conclude that in vivo, repriming occurs ∼70× less frequently on the leading versus lagging strands, and that DNA replication inE. coliis effectively semidiscontinuous.

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Hernandez, Alfredo J., Seung-Joo Lee, and Charles C. Richardson. "Primer release is the rate-limiting event in lagging-strand synthesis mediated by the T7 replisome." Proceedings of the National Academy of Sciences 113, no. 21 (2016): 5916–21. http://dx.doi.org/10.1073/pnas.1604894113.

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DNA replication occurs semidiscontinuously due to the antiparallel DNA strands and polarity of enzymatic DNA synthesis. Although the leading strand is synthesized continuously, the lagging strand is synthesized in small segments designated Okazaki fragments. Lagging-strand synthesis is a complex event requiring repeated cycles of RNA primer synthesis, transfer to the lagging-strand polymerase, and extension effected by cooperation between DNA primase and the lagging-strand polymerase. We examined events controlling Okazaki fragment initiation using the bacteriophage T7 replication system. Primer utilization by T7 DNA polymerase is slower than primer formation. Slow primer release from DNA primase allows the polymerase to engage the complex and is followed by a slow primer handoff step. The T7 single-stranded DNA binding protein increases primer formation and extension efficiency but promotes limited rounds of primer extension. We present a model describing Okazaki fragment initiation, the regulation of fragment length, and their implications for coordinated leading- and lagging-strand DNA synthesis.

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Bartoszek, Krzysztof, and Wojciech Bartoszek. "On the time behaviour of Okazaki fragments." Journal of Applied Probability 43, no. 02 (2006): 500–509. http://dx.doi.org/10.1017/s0021900200001789.

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We find explicit analytical formulae for the time dependence of the probability of the number of Okazaki fragments produced during the process of DNA replication. This extends a result of Cowan on the asymptotic probability distribution of these fragments.

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Bartoszek, Krzysztof, and Wojciech Bartoszek. "On the time behaviour of Okazaki fragments." Journal of Applied Probability 43, no. 2 (2006): 500–509. http://dx.doi.org/10.1239/jap/1152413737.

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We find explicit analytical formulae for the time dependence of the probability of the number of Okazaki fragments produced during the process of DNA replication. This extends a result of Cowan on the asymptotic probability distribution of these fragments.

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9

Henneke, Ghislaine. "In vitro reconstitution of RNA primer removal in Archaea reveals the existence of two pathways." Biochemical Journal 447, no. 2 (2012): 271–80. http://dx.doi.org/10.1042/bj20120959.

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Using model DNA substrates and purified recombinant proteins from Pyrococcus abyssi, I have reconstituted the enzymatic reactions involved in RNA primer elimination in vitro. In my dual-labelled system, polymerase D performed efficient strand displacement DNA synthesis, generating 5′-RNA flaps which were subsequently released by Fen1, before ligation by Lig1. In this pathway, the initial cleavage event by RNase HII facilitated RNA primer removal of Okazaki fragments. In addition, I have shown that polymerase B was able to displace downstream DNA strands with a single ribonucleotide at the 5′-end, a product resulting from a single cut in the RNA initiator by RNase HII. After RNA elimination, the combined activities of strand displacement DNA synthesis by polymerase B and flap cleavage by Fen1 provided a nicked substrate for ligation by Lig1. The unique specificities of Okazaki fragment maturation enzymes and replicative DNA polymerases strongly support the existence of two pathways in the resolution of RNA fragments.

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Kang, Ho-Young, Eunjoo Choi, Sung-Ho Bae, et al. "Genetic Analyses of Schizosaccharomyces pombe dna2+ Reveal That Dna2 Plays an Essential Role in Okazaki Fragment Metabolism." Genetics 155, no. 3 (2000): 1055–67. http://dx.doi.org/10.1093/genetics/155.3.1055.

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Abstract In this report, we investigated the phenotypes caused by temperature-sensitive (ts) mutant alleles of dna2+ of Schizosaccharomyces pombe, a homologue of DNA2 of budding yeast, in an attempt to further define its function in vivo with respect to lagging-strand synthesis during the S-phase of the cell cycle. At the restrictive temperature, dna2 (ts) cells arrested at late S-phase but were unaffected in bulk DNA synthesis. Moreover, they exhibited aberrant mitosis when combined with checkpoint mutations, in keeping with a role for Dna2 in Okazaki fragment maturation. Similarly, spores in which dna2+ was disrupted duplicated their DNA content during germination and also arrested at late S-phase. Inactivation of dna2+ led to chromosome fragmentation strikingly similar to that seen when cdc17+, the DNA ligase I gene, is inactivated. The temperature-dependent lethality of dna2 (ts) mutants was suppressed by overexpression of genes encoding subunits of polymerase δ (cdc1+ and cdc27+), DNA ligase I (cdc17+), and Fen-1 (rad2+). Each of these gene products plays a role in the elongation or maturation of Okazaki fragments. Moreover, they all interacted with S. pombe Dna2 in a yeast two-hybrid assay, albeit to different extents. On the basis of these results, we conclude that dna2+ plays a direct role in the Okazaki fragment elongation and maturation. We propose that dna2+ acts as a central protein to form a complex with other proteins required to coordinate the multienzyme process for Okazaki fragment elongation and maturation.

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Vaitsiankova, Alina, Kamila Burdova, Margarita Sobol, et al. "PARP inhibition impedes the maturation of nascent DNA strands during DNA replication." Nature Structural & Molecular Biology 29, no. 4 (2022): 329–38. http://dx.doi.org/10.1038/s41594-022-00747-1.

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AbstractPoly(ADP-ribose) polymerase 1 (PARP1) is implicated in the detection and processing of unligated Okazaki fragments and other DNA replication intermediates, highlighting such structures as potential sources of genome breakage induced by PARP inhibition. Here, we show that PARP1 activity is greatly elevated in chicken and human S phase cells in which FEN1 nuclease is genetically deleted and is highest behind DNA replication forks. PARP inhibitor reduces the integrity of nascent DNA strands in both wild-type chicken and human cells during DNA replication, and does so in FEN1−/− cells to an even greater extent that can be detected as postreplicative single-strand nicks or gaps. Collectively, these data show that PARP inhibitors impede the maturation of nascent DNA strands during DNA replication, and implicate unligated Okazaki fragments and other nascent strand discontinuities in the cytotoxicity of these compounds.

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Soto, Ana Maria, William H. Gmeiner, and Luis A. Marky. "Energetic and Conformational Contributions to the Stability of Okazaki Fragments†." Biochemistry 41, no. 21 (2002): 6842–49. http://dx.doi.org/10.1021/bi025715o.

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13

Yanga, Wenchao, and Xinhui Lib. "Next-generation sequencing of Okazaki fragments extracted from Saccharomyces cerevisiae." FEBS Letters 587, no. 15 (2013): 2441–47. http://dx.doi.org/10.1016/j.febslet.2013.06.014.

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Budd, M. E., and J. L. Campbell. "A yeast replicative helicase, Dna2 helicase, interacts with yeast FEN-1 nuclease in carrying out its essential function." Molecular and Cellular Biology 17, no. 4 (1997): 2136–42. http://dx.doi.org/10.1128/mcb.17.4.2136.

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We have recently described a new helicase, the Dna2 helicase, that is essential for yeast DNA replication. We now show that the yeast FEN-1 (yFEN-1) nuclease interacts genetically and biochemically with Dna2 helicase. FEN-1 is implicated in DNA replication and repair in yeast, and the mammalian homolog of yFEN-1 (DNase IV, FEN-1, or MF1) participates in Okazaki fragment maturation. Overproduction of yFEN-1, encoded by RAD27/RTH1, suppresses the temperature-sensitive growth of dna2-1 mutants. Overproduction of Dna2 suppresses the rad27/rth1 delta temperature-sensitive growth defect. dna2-1 rad27/rth1 delta double mutants are inviable, indicating that the mutations are synthetically lethal. The genetic interactions are likely due to direct physical interaction between the two proteins, since both epitope-tagged yFEN-1 and endogenous yFEN-1 coimmunopurify with tagged Dna2. The simplest interpretation of these data is that one of the roles of Dna2 helicase is associated with processing of Okazaki fragments.

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Bae, Sung-Ho, Kwang-Hee Bae, Jung-Ae Kim, and Yeon-Soo Seo. "RPA governs endonuclease switching during processing of Okazaki fragments in eukaryotes." Nature 412, no. 6845 (2001): 456–61. http://dx.doi.org/10.1038/35086609.

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Salazar, Miguel, James J. Champoux, and Brian R. Reid. "Sugar conformations at hybrid duplex junctions in HIV-1 and Okazaki fragments." Biochemistry 32, no. 3 (1993): 739–44. http://dx.doi.org/10.1021/bi00054a002.

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Matsunaga, Fujihiko, Cédric Norais, Patrick Forterre, and Hannu Myllykallio. "Identification of short ‘eukaryotic’ Okazaki fragments synthesized from a prokaryotic replication origin." EMBO reports 4, no. 2 (2003): 154–58. http://dx.doi.org/10.1038/sj.embor.embor732.

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18

Wang, T. C. V., and S. H. Chen. "Okazaki DNA Fragments Contain Equal Amounts of Lagging-Strand and Leading-Strand Sequences." Biochemical and Biophysical Research Communications 198, no. 3 (1994): 844–49. http://dx.doi.org/10.1006/bbrc.1994.1120.

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19

Qiu, Junzhuan, Ying Qian, Peter Frank, Ulrike Wintersberger, and Binghui Shen. "Saccharomyces cerevisiae RNase H(35) Functions in RNA Primer Removal during Lagging-Strand DNA Synthesis, Most Efficiently in Cooperation with Rad27 Nuclease." Molecular and Cellular Biology 19, no. 12 (1999): 8361–71. http://dx.doi.org/10.1128/mcb.19.12.8361.

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ABSTRACT Correct removal of RNA primers of Okazaki fragments during lagging-strand DNA synthesis is a critical process for the maintenance of genome integrity. Disturbance of this process has severe mutagenic consequences and could contribute to the development of cancer. The role of the mammalian nucleases RNase HI and FEN-1 in RNA primer removal has been substantiated by several studies. Recently, RNase H(35), the Saccharomyces cerevisiae homologue of mammalian RNase HI, was identified and its possible role in DNA replication was proposed (P. Frank, C. Braunshofer-Reiter, and U. Wintersberger, FEBS Lett. 421:23–26, 1998). This led to the possibility of moving to the genetically powerful yeast system for studying the homologues of RNase HI and FEN-1, i.e., RNase H(35) and Rad27p, respectively. In this study, we have biochemically defined the substrate specificities and the cooperative as well as independent cleavage mechanisms ofS. cerevisiae RNase H(35) and Rad27 nuclease by using Okazaki fragment model substrates. We have also determined the additive and compensatory pathological effects of gene deletion and overexpression of these two enzymes. Furthermore, the mutagenic consequences of the nuclease deficiencies have been analyzed. Based on our findings, we suggest that three alternative RNA primer removal pathways of different efficiencies involve RNase H(35) and Rad27 nucleases in yeast.

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Beattie, Thomas R., and Stephen D. Bell. "The role of the DNA sliding clamp in Okazaki fragment maturation in archaea and eukaryotes." Biochemical Society Transactions 39, no. 1 (2011): 70–76. http://dx.doi.org/10.1042/bst0390070.

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Efficient processing of Okazaki fragments generated during discontinuous lagging-strand DNA replication is critical for the maintenance of genome integrity. In eukaryotes, a number of enzymes co-ordinate to ensure the removal of initiating primers from the 5′-end of each fragment and the generation of a covalently linked daughter strand. Studies in eukaryotic systems have revealed that the co-ordination of DNA polymerase δ and FEN-1 (Flap Endonuclease 1) is sufficient to remove the majority of primers. Other pathways such as that involving Dna2 also operate under certain conditions, although, notably, Dna2 is not universally conserved between eukaryotes and archaea, unlike the other core factors. In addition to the catalytic components, the DNA sliding clamp, PCNA (proliferating-cell nuclear antigen), plays a pivotal role in binding and co-ordinating these enzymes at sites of lagging-strand replication. Structural studies in eukaryotic and archaeal systems have revealed that PCNA-binding proteins can adopt different conformations when binding PCNA. This conformational malleability may be key to the co-ordination of these enzymes' activities.

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Kellner, Vanessa, and Duncan J. Smith. "Hold on Tight: Lagging-Strand DNA Polymerases Synthesize Multiple Okazaki Fragments without Letting Go." Molecular Cell 80, no. 1 (2020): 6–8. http://dx.doi.org/10.1016/j.molcel.2020.09.010.

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Parenteau, Julie, and Raymund J. Wellinger. "Accumulation of Single-Stranded DNA and Destabilization of Telomeric Repeats in Yeast Mutant Strains Carrying a Deletion of RAD27." Molecular and Cellular Biology 19, no. 6 (1999): 4143–52. http://dx.doi.org/10.1128/mcb.19.6.4143.

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ABSTRACT The Saccharomyces cerevisiae RAD27 gene encodes the yeast homologue of the mammalian FEN-1 nuclease, a protein that is thought to be involved in the processing of Okazaki fragments during DNA lagging-strand synthesis. One of the predicted DNA lesions occurring in rad27 strains is the presence of single-stranded DNA of the template strand for lagging-strand synthesis. We examined this prediction by analyzing the terminal DNA structures generated during telomere replication in rad27strains. The lengths of the telomeric repeat tracts were found to be destabilized in rad27 strains, indicating that naturally occurring direct repeats are subject to tract expansions and contractions in such strains. Furthermore, abnormally high levels of single-stranded DNA of the templating strand for lagging-strand synthesis were observed in rad27 cells. Overexpression of Dna2p in wild-type cells also yielded single-stranded DNA regions on telomeric DNA and caused a cell growth arrest phenotype virtually identical to that seen for rad27 cells grown at the restrictive temperature. Furthermore, overexpression of the yeast exonuclease Exo1p alleviated the growth arrest induced by both conditions, overexpression of Dna2p and incubation of rad27cells at 37°C. However, the telomere heterogeneity and the appearance of single-stranded DNA are not prevented by the overexpression of Exo1p in these strains, suggesting that this nuclease is not simply redundant with Rad27p. Our data thus provide in vivo evidence for the types of DNA lesions predicted to occur when lagging-strand synthesis is deficient and suggest that Dna2p and Rad27p collaborate in the processing of Okazaki fragments.

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Huang, L., Y. Kim, J. J. Turchi, and R. A. Bambara. "Structure-specific cleavage of the RNA primer from Okazaki fragments by calf thymus RNase HI." Journal of Biological Chemistry 269, no. 41 (1994): 25922–27. http://dx.doi.org/10.1016/s0021-9258(18)47334-6.

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Murante, Richard S., Jeffrey A. Rumbaugh, Carole J. Barnes, J. Russell Norton, and Robert A. Bambara. "Calf RTH-1 Nuclease Can Remove the Initiator RNAs of Okazaki Fragments by Endonuclease Activity." Journal of Biological Chemistry 271, no. 42 (1996): 25888–97. http://dx.doi.org/10.1074/jbc.271.42.25888.

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25

Nguyen, Hai Dang, Jordan Becker, Yee Mon Thu, et al. "Unligated Okazaki Fragments Induce PCNA Ubiquitination and a Requirement for Rad59-Dependent Replication Fork Progression." PLoS ONE 8, no. 6 (2013): e66379. http://dx.doi.org/10.1371/journal.pone.0066379.

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Liu, Beiyu, Jianyang Wang, Gokben Yildirir, and Paul T. Englund. "TbPIF5 Is a Trypanosoma brucei Mitochondrial DNA Helicase Involved in Processing of Minicircle Okazaki Fragments." PLoS Pathogens 5, no. 9 (2009): e1000589. http://dx.doi.org/10.1371/journal.ppat.1000589.

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Rossi, Marie L., Jason E. Pike, Wensheng Wang, Peter M. J. Burgers, Judith L. Campbell, and Robert A. Bambara. "Pif1 Helicase Directs Eukaryotic Okazaki Fragments toward the Two-nuclease Cleavage Pathway for Primer Removal." Journal of Biological Chemistry 283, no. 41 (2008): 27483–93. http://dx.doi.org/10.1074/jbc.m804550200.

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Cowan, Richard, and S. N. Chiu. "A stochastic model of fragment formation when DNA replicates." Journal of Applied Probability 31, no. 2 (1994): 301–8. http://dx.doi.org/10.2307/3215025.

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The double-stranded molecule, DNA, has the unique property of replication and, because of this, it is the central molecule of life. The mechanism of replication for each single strand is intricate, involving enzymes which move along each of the single strands building a complementary copy. At the frontier of this action, the events have a strong stochastic character due to the random location on the DNA of key ‘sites' where copying commences. A model of this process is analysed. The central problem of interest is the mean length of certain ‘islands' of newly replicated DNA developed at the randomly located ‘sites'. These islands, which have been observed experimentally, are called Okazaki fragments.

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Cowan, Richard, and S. N. Chiu. "A stochastic model of fragment formation when DNA replicates." Journal of Applied Probability 31, no. 02 (1994): 301–8. http://dx.doi.org/10.1017/s0021900200044843.

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The double-stranded molecule, DNA, has the unique property of replication and, because of this, it is the central molecule of life. The mechanism of replication for each single strand is intricate, involving enzymes which move along each of the single strands building a complementary copy. At the frontier of this action, the events have a strong stochastic character due to the random location on the DNA of key ‘sites' where copying commences. A model of this process is analysed. The central problem of interest is the mean length of certain ‘islands' of newly replicated DNA developed at the randomly located ‘sites'. These islands, which have been observed experimentally, are called Okazaki fragments.

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Bellizzi, Dina, M. Adele Losso, and Vittorio Sgaramella. "A model for the involvement of Okazaki fragments maturation in the expansion of short tandem repeats." Gene 276, no. 1-2 (2001): 153–59. http://dx.doi.org/10.1016/s0378-1119(01)00642-4.

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Kahli, Malik, Joseph S. Osmundson, Rani Yeung, and Duncan J. Smith. "Processing of eukaryotic Okazaki fragments by redundant nucleases can be uncoupled from ongoing DNA replicationin vivo." Nucleic Acids Research 47, no. 4 (2018): 1814–22. http://dx.doi.org/10.1093/nar/gky1242.

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Bartos, Jeremy D., Wensheng Wang, Jason E. Pike, and Robert A. Bambara. "Mechanisms by Which Bloom Protein Can Disrupt Recombination Intermediates of Okazaki Fragment Maturation." Journal of Biological Chemistry 281, no. 43 (2006): 32227–39. http://dx.doi.org/10.1074/jbc.m606310200.

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Bloom syndrome is a familial genetic disorder associated with sunlight sensitivity and a high predisposition to cancers. The mutated gene, Bloom protein (BLM), encodes a DNA helicase that functions in genome maintenance via roles in recombination repair and resolution of recombination structures. We designed substrates representing illegitimate recombination intermediates formed when a displaced DNA flap generated during maturation of Okazaki fragments escapes cleavage by flap endonuclease-1 and anneals to a complementary ectopic DNA site. Results show that displaced, replication protein A (RPA)-coated flaps could readily bind and ligate at the complementary site to initiate recombination. RPA also displayed a strand-annealing activity that hastens the rate of recombination intermediate formation. BLM helicase activity could directly disrupt annealing at the ectopic site and promote flap endonuclease-1 cleavage. Additionally, BLM has its own strand-annealing and strand-exchange activities. RPA inhibited the BLM strand-annealing activity, thereby promoting helicase activity and complex dissolution. BLM strand exchange could readily dissociate invading flaps, e.g. in a D-loop, if the exchange step did not involve annealing of RPA-coated strands. Use of ATP to activate the helicase function did not aid flap displacement by exchange, suggesting that this is a helicase-independent mechanism of complex dissociation. When RPA could bind, it displayed its own strand-exchange activity. We interpret these results to explain how BLM is well equipped to deal with alternative recombination intermediate structures.

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Muzi-Falconi, Marco, Michele Giannattasio, Marco Foiani, and Paolo Plevani. "The DNA Polymerase _-Primase Complex: Multiple Functions and Interactions." Scientific World JOURNAL 3 (2003): 21–33. http://dx.doi.org/10.1100/tsw.2003.05.

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DNA polymerase _ (pol _) holds a special position among the growing family of eukaryotic DNA polymerases. In fact, pol _ is associated with DNA primase to form a four subunit complex and, as a consequence, is the only enzyme able to start DNA synthesis de novo. Because of this peculiarity the major role of the DNA polymerase _-primase complex (pol-prim) is in the initiation of DNA replication at chromosomal origins and in the discontinuous synthesis of Okazaki fragments on the lagging strand of the replication fork. However, pol-prim seems to play additional roles in other complex cellular processes, such as the response to DNA damage, telomere maintenance, and the epigenetic control of higher order chromatin assembly.

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Hanzlikova, Hana, Ilona Kalasova, Annie A. Demin, Lewis E. Pennicott, Zuzana Cihlarova, and Keith W. Caldecott. "The Importance of Poly(ADP-Ribose) Polymerase as a Sensor of Unligated Okazaki Fragments during DNA Replication." Molecular Cell 71, no. 2 (2018): 319–31. http://dx.doi.org/10.1016/j.molcel.2018.06.004.

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Levikova, Maryna, and Petr Cejka. "TheSaccharomyces cerevisiaeDna2 can function as a sole nuclease in the processing of Okazaki fragments in DNA replication." Nucleic Acids Research 43, no. 16 (2015): 7888–97. http://dx.doi.org/10.1093/nar/gkv710.

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Choe, Wonchae, Martin Budd, Osamu Imamura, Laura Hoopes, and Judith L. Campbell. "Dynamic Localization of an Okazaki Fragment Processing Protein Suggests a Novel Role in Telomere Replication." Molecular and Cellular Biology 22, no. 12 (2002): 4202–17. http://dx.doi.org/10.1128/mcb.22.12.4202-4217.2002.

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ABSTRACT We have found that the Dna2 helicase-nuclease, thought to be involved in maturation of Okazaki fragments, is a component of telomeric chromatin. We demonstrate a dynamic localization of Dna2p to telomeres that suggests a dual role for Dna2p, one in telomere replication and another, unknown function, perhaps in telomere capping. Both chromatin immunoprecipitation (ChIP) and immunofluorescence show that Dna2p associates with telomeres but not bulk chromosomal DNA in G1 phase, when there is no telomere replication and the telomere is transcriptionally silenced. In S phase, there is a dramatic redistribution of Dna2p from telomeres to sites throughout the replicating chromosomes. Dna2p is again localized to telomeres in late S, where it remains through G2 and until the next S phase. Telomeric localization of Dna2p required Sir3p, since the amount of Dna2p found at telomeres by two different assays, one-hybrid and ChIP, is severely reduced in strains lacking Sir3p. The Dna2p is also distributed throughout the nucleus in cells growing in the presence of double-strand-break-inducing agents such as bleomycin. Finally, we show that Dna2p is functionally required for telomerase-dependent de novo telomere synthesis and also participates in telomere lengthening in mutants lacking telomerase.

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van Schendel, Robin, Ron Romeijn, Helena Buijs та Marcel Tijsterman. "Preservation of lagging strand integrity at sites of stalled replication by Pol α-primase and 9-1-1 complex". Science Advances 7, № 21 (2021): eabf2278. http://dx.doi.org/10.1126/sciadv.abf2278.

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During genome duplication, the replication fork encounters a plethora of obstacles in the form of damaged bases, DNA–cross-linked proteins, and secondary structures. How cells protect DNA integrity at sites of stalled replication is currently unknown. Here, by engineering “primase deserts” into the Caenorhabditis elegans genome close to replication-impeding G-quadruplexes, we show that de novo DNA synthesis downstream of the blocked fork suppresses DNA loss. We next identify the pol α-primase complex to limit deletion mutagenesis, a conclusion substantiated by whole-genome analysis of animals carrying mutated POLA2/DIV-1. We subsequently identify a new role for the 9-1-1 checkpoint clamp in protecting Okazaki fragments from resection by EXO1. Together, our results provide a mechanistic model for controlling the fate of replication intermediates at sites of stalled replication.

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CSUKA, ILDIKO, and GASPAR BANFALVI. "Analysis of 5′-Termini of Early Intermediates of Okazaki Fragments Accumulated in Thymocytes after Emetine Treatment of Mice." DNA and Cell Biology 16, no. 8 (1997): 979–84. http://dx.doi.org/10.1089/dna.1997.16.979.

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Merrill, Bradley J., and Connie Holm. "The RAD52 Recombinational Repair Pathway is Essential in pol30 (PCNA) Mutants That Accumulate Small Single-Stranded DNA Fragments During DNA Synthesis." Genetics 148, no. 2 (1998): 611–24. http://dx.doi.org/10.1093/genetics/148.2.611.

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Abstract To identify in vivo pathways that compensate for impaired proliferating cell nuclear antigen (PCNA or Pol30p in yeast) activity, we performed a synthetic lethal screen with the yeast pol30-104 mutation. We identified nine mutations that display synthetic lethality with pol30-104; three mutations affected the structural gene for the large subunit of replication factor C (rfc1), which loads PCNA onto DNA, and six mutations affected three members of the RAD52 epistasis group for DNA recombinational repair (rad50, rad52, and rad57). We also found that pol30-104 displayed synthetic lethality with mutations in other members of the RAD52 epistasis group (rad51 and rad54), but not with mutations in members of the RAD3 nor the RAD6 epistasis group. Analysis of nine different pol30 mutations shows that the requirement for the RAD52 pathway is correlated with a DNA replication defect but not with the relative DNA repair defect caused by pol30 mutations. In addition, mutants that require RAD52 for viability (pol30-100, pol30-104, rfc1-1 and rth1Δ) accumulate small single-stranded DNA fragments during DNA replication in vivo. Taken together, these data suggest that the RAD52 pathway is required when there are defects in the maturation of Okazaki fragments.

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Le Laz, Sébastien, Audrey Le Goaziou, and Ghislaine Henneke. "Structure-Specific Nuclease Activities of Pyrococcus abyssi RNase HII." Journal of Bacteriology 192, no. 14 (2010): 3689–98. http://dx.doi.org/10.1128/jb.00268-10.

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ABSTRACT Faithful DNA replication involves the removal of RNA residues from genomic DNA prior to the ligation of nascent DNA fragments in all living organisms. Because the physiological roles of archaeal type 2 RNase H are not fully understood, the substrate structure requirements for the detection of RNase H activity need further clarification. Biochemical characterization of a single RNase H detected within the genome of Pyrococcus abyssi showed that this type 2 RNase H is an Mg- and alkaline pH-dependent enzyme. PabRNase HII showed RNase activity and acted as a specific endonuclease on RNA-DNA/DNA duplexes. This specific cleavage, 1 nucleotide upstream of the RNA-DNA junction, occurred on a substrate in which RNA initiators had to be fully annealed to the cDNA template. On the other hand, a 5′ RNA flap Okazaki fragment intermediate impaired PabRNase HII endonuclease activity. Furthermore, introduction of mismatches into the RNA portion near the RNA-DNA junction decreased both the specificity and the efficiency of cleavage by PabRNase HII. Additionally, PabRNase HII could cleave a single ribonucleotide embedded in a double-stranded DNA. Our data revealed PabRNase HII as a dual-function enzyme likely required for the completion of DNA replication and DNA repair.

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Pohjanpelto, P., and E. Hölttä. "Phosphorylation of Okazaki-like DNA fragments in mammalian cells and role of polyamines in the processing of this DNA." EMBO Journal 15, no. 5 (1996): 1193–200. http://dx.doi.org/10.1002/j.1460-2075.1996.tb00458.x.

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Larsen, Elisabeth, Christine Gran, Barbro Elisabet Sæther, Erling Seeberg, and Arne Klungland. "Proliferation Failure and Gamma Radiation Sensitivity of Fen1 Null Mutant Mice at the Blastocyst Stage." Molecular and Cellular Biology 23, no. 15 (2003): 5346–53. http://dx.doi.org/10.1128/mcb.23.15.5346-5353.2003.

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ABSTRACT Flap endonuclease 1 (FEN1) has been shown to remove 5′ overhanging flap intermediates during base excision repair and to process the 5′ ends of Okazaki fragments during lagging-strand DNA replication in vitro. To assess the in vivo role of the mammalian enzyme in repair and replication, we used a gene-targeting approach to generate mice lacking a functional Fen1 gene. Heterozygote animals appear normal, whereas complete depletion of FEN1 causes early embryonic lethality. Fen1−/− blastocysts fail to form inner cell mass during cellular outgrowth, and a complete inactivation of DNA synthesis in giant cells of blastocyst outgrowth was observed. Exposure of Fen1−/− blastocysts to gamma radiation caused extensive apoptosis, implying an essential role for FEN1 in the repair of radiation-induced DNA damage in vivo. Our data thus provide in vivo evidence for an essential function of FEN1 in DNA repair, as well as in DNA replication.

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Prigent, C., M. S. Satoh, G. Daly, D. E. Barnes, and T. Lindahl. "Aberrant DNA repair and DNA replication due to an inherited enzymatic defect in human DNA ligase I." Molecular and Cellular Biology 14, no. 1 (1994): 310–17. http://dx.doi.org/10.1128/mcb.14.1.310.

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Two missense mutations in different alleles of the DNA ligase I gene have been described in a patient (46BR) with immunodeficiencies and cellular hypersensitivity to DNA-damaging agents. One of the mutant alleles produces an inactive protein, while the other encodes an enzyme with some residual activity. A subline of identical phenotype that is homozygous (or hemizygous) for the mutant allele encoding this partially active enzyme has facilitated characterization of the enzymatic defect in 46BR. This subline retains only 3 to 5% of normal DNA ligase I activity. The intermediates in the ligation reaction, DNA ligase I-AMP and nicked DNA-AMP, accumulate in vitro and in vivo. The defect of the 46BR enzyme lies primarily in conversion of nicked DNA-AMP into the final ligated DNA product. Assays of DNA repair in 46BR cell extracts and of DNA replication in permeabilized cells have clarified functional roles of DNA ligase I. The initial rate of ligation of Okazaki fragments during DNA replication is apparently normal in 46BR cells, but 25 to 30% of the fragments remain in low-molecular-weight form for prolonged times. DNA base excision repair by 46BR cell extracts shows a delay in ligation and an anomalously long repair patch size that is reduced upon addition of purified normal DNA ligase I.

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Prigent, C., M. S. Satoh, G. Daly, D. E. Barnes, and T. Lindahl. "Aberrant DNA repair and DNA replication due to an inherited enzymatic defect in human DNA ligase I." Molecular and Cellular Biology 14, no. 1 (1994): 310–17. http://dx.doi.org/10.1128/mcb.14.1.310-317.1994.

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Two missense mutations in different alleles of the DNA ligase I gene have been described in a patient (46BR) with immunodeficiencies and cellular hypersensitivity to DNA-damaging agents. One of the mutant alleles produces an inactive protein, while the other encodes an enzyme with some residual activity. A subline of identical phenotype that is homozygous (or hemizygous) for the mutant allele encoding this partially active enzyme has facilitated characterization of the enzymatic defect in 46BR. This subline retains only 3 to 5% of normal DNA ligase I activity. The intermediates in the ligation reaction, DNA ligase I-AMP and nicked DNA-AMP, accumulate in vitro and in vivo. The defect of the 46BR enzyme lies primarily in conversion of nicked DNA-AMP into the final ligated DNA product. Assays of DNA repair in 46BR cell extracts and of DNA replication in permeabilized cells have clarified functional roles of DNA ligase I. The initial rate of ligation of Okazaki fragments during DNA replication is apparently normal in 46BR cells, but 25 to 30% of the fragments remain in low-molecular-weight form for prolonged times. DNA base excision repair by 46BR cell extracts shows a delay in ligation and an anomalously long repair patch size that is reduced upon addition of purified normal DNA ligase I.

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Gmeiner, W. H. "NMR Spectroscopy as A Tool to Investigate the Structural Basis of Anticancer Drugs." Current Medicinal Chemistry 5, no. 2 (1998): 115–35. http://dx.doi.org/10.2174/0929867305666220314202136.

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NMR spectroscopy has been shown to be useful in determining the structures of nucleic acid fragments in solution. Over the last several years NMR spectroscopy, in conjunction with restrained molecular dynamics, has been employed to understand the 3D structures of a number of anticancer drugs and to rationalize their DNA binding behavior. In this review we address the methodologies used most frequently to determine nucleic acid structures in solution. In subsequent sections, we examine how these methods have been applied to rationalize the activities of a number of anticancer agents that target duplex DNA such as cisplatin, bleomycin and calicheamicin. Non-duplex DNA and RNA also represent interesting nucleic acid targets for anticancer drug design and applications of solution NMR spectroscopy to understanding the structures of these types of molecules (e.g. Okazaki fragments, DNA tetraplexes) are also reviewed. In the final sections, advances in NMR methodologies (e.g. linear prediction, superconducting probes) that are likely to impact the research conducted in this area are reviewed. The success of NMR spectroscopy in understanding the structural basis for clinically useful anticancer drugs bodes well for future applications of this methodology not only in rationalization of existing biological activity, but in the design of novel agents that will be useful in treating neoplastic disease.

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Levin, David S., Allison E. McKenna, Teresa A. Motycka, Yoshihiro Matsumoto, and Alan E. Tomkinson. "Interaction between PCNA and DNA ligase I is critical for joining of Okazaki fragments and long-patch base-excision repair." Current Biology 10, no. 15 (2000): 919—S2. http://dx.doi.org/10.1016/s0960-9822(00)00619-9.

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Gloor, Jason W., Lata Balakrishnan, Judith L. Campbell, and Robert A. Bambara. "Biochemical analyses indicate that binding and cleavage specificities define the ordered processing of human Okazaki fragments by Dna2 and FEN1." Nucleic Acids Research 40, no. 14 (2012): 6774–86. http://dx.doi.org/10.1093/nar/gks388.

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Shapiro, Adam B., Ann E. Eakin, Grant K. Walkup, and Olga Rivin. "A High-Throughput Fluorescence Resonance Energy Transfer-Based Assay for DNA Ligase." Journal of Biomolecular Screening 16, no. 5 (2011): 486–93. http://dx.doi.org/10.1177/1087057111398295.

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DNA ligase is the enzyme that catalyzes the formation of the backbone phosphodiester bond between the 5′-PO4 and 3′-OH of adjacent DNA nucleotides at single-stranded nicks. These nicks occur between Okazaki fragments during replication of the lagging strand of the DNA as well as during DNA repair and recombination. As essential enzymes for DNA replication, the NAD+-dependent DNA ligases of pathogenic bacteria are potential targets for the development of antibacterial drugs. For the purposes of drug discovery, a high-throughput assay for DNA ligase activity is invaluable. This article describes a straightforward, fluorescence resonance energy transfer–based DNA ligase assay that is well suited for high-throughput screening for DNA ligase inhibitors as well as for use in enzyme kinetics studies. Its use is demonstrated for measurement of the steady-state kinetic constants of Haemophilus influenzae NAD+-dependent DNA ligase and for measurement of the potency of an inhibitor of this enzyme.

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Kokoska, Robert J., Lela Stefanovic, Hiep T. Tran, Michael A. Resnick, Dmitry A. Gordenin та Thomas D. Petes. "Destabilization of Yeast Micro- and Minisatellite DNA Sequences by Mutations Affecting a Nuclease Involved in Okazaki Fragment Processing (rad27) and DNA Polymerase δ (pol3-t)". Molecular and Cellular Biology 18, № 5 (1998): 2779–88. http://dx.doi.org/10.1128/mcb.18.5.2779.

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ABSTRACT We examined the effects of mutations in the Saccharomyces cerevisiae RAD27 (encoding a nuclease involved in the processing of Okazaki fragments) and POL3 (encoding DNA polymerase δ) genes on the stability of a minisatellite sequence (20-bp repeats) and microsatellites (1- to 8-bp repeat units). Both therad27 and pol3-t mutations destabilized both classes of repeats, although the types of tract alterations observed in the two mutant strains were different. The tract alterations observed in rad27 strains were primarily additions, and those observed in pol3-t strains were primarily deletions. Measurements of the rates of repetitive tract alterations in strains with both rad27 and pol3-t indicated that the stimulation of microsatellite instability by rad27 was reduced by the effects of the pol3-t mutation. We also found that rad27 and pol3-01 (an allele carrying a mutation in the “proofreading” exonuclease domain of DNA polymerase δ) mutations were synthetically lethal.

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Denis, D., and P. A. Bullock. "Primer-DNA formation during simian virus 40 DNA replication in vitro." Molecular and Cellular Biology 13, no. 5 (1993): 2882–90. http://dx.doi.org/10.1128/mcb.13.5.2882.

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Studies of simian virus 40 (SV40) DNA replication in vitro have identified a small (approximately 30-nucleotide) RNA-DNA hybrid species termed primer-DNA. Initial experiments indicated that T antigen and the polymerase alpha-primase complex are required to form primer-DNA. Proliferating cell nuclear antigen, and presumably proliferating cell nuclear antigen-dependent polymerases, is not needed to form this species. Herein, we present an investigation of the stages at which primer-DNA functions during SV40 DNA replication in vitro. Hybridization studies indicate that primer-DNA is initially formed in the origin region and is subsequently synthesized in regions distal to the origin. At all time points, primer-DNA is synthesized from templates for lagging-strand DNA replication. These studies indicate that primer-DNA functions during both initiation and elongation stages of SV40 DNA synthesis. Results of additional experiments suggesting a precursor-product relationship between formation of primer-DNA and Okazaki fragments are presented.

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Journal articles on the topic 'Okazaki fragments'

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