scholarly journals The architecture of an Okazaki fragment-processing holoenzyme from the archaeon Sulfolobus solfataricus

2015 ◽  
Vol 465 (2) ◽  
pp. 239-245 ◽  
Author(s):  
Giuseppe Cannone ◽  
Yuli Xu ◽  
Thomas R. Beattie ◽  
Stephen D. Bell ◽  
Laura Spagnolo
Keyword(s):  
Electron Microscopy ◽  
Sulfolobus Solfataricus ◽  
Model Organism ◽  
Okazaki Fragment ◽  
Okazaki Fragment Processing ◽  
Okazaki Fragment Maturation

Electron microscopy confirms the mechanism adopted by PCNA to concert the action of multiple enzymes during Okazaki fragment maturation in the model organism Sulfolobus solfataricus.

scholarly journals RNase HIII Is Important for Okazaki Fragment Processing inBacillus subtilis

2019 ◽  
Vol 201 (7) ◽  
Author(s):  
Justin R. Randall ◽  
Taylor M. Nye ◽  
Katherine J. Wozniak ◽  
Lyle A. Simmons
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerase I ◽  
Okazaki Fragment ◽  
Okazaki Fragments ◽  
Okazaki Fragment Processing ◽  
Rnase Hi ◽  
Okazaki Fragment Maturation ◽  
Polymerase I

ABSTRACTRNA-DNA hybrids are common in chromosomal DNA. Persistent RNA-DNA hybrids result in replication fork stress, DNA breaks, and neurological disorders in humans. During replication, Okazaki fragment synthesis relies on frequent RNA primer placement, providing one of the most prominent forms of covalent RNA-DNA strandsin vivo. The mechanism of Okazaki fragment maturation, which involves RNA removal and subsequent DNA replacement, in bacteria lacking RNase HI remains unclear. In this work, we reconstituted repair of a linear model Okazaki fragmentin vitrousing purified recombinant enzymes fromBacillus subtilis. We showed that RNase HII and HIII are capable of incision on Okazaki fragmentsin vitroand that both enzymes show mild stimulation by single-stranded DNA binding protein (SSB). We also showed that RNase HIII and DNA polymerase I provide the primary pathway for Okazaki fragment maturationin vitro. Furthermore, we found that YpcP is a 5′ to 3′ nuclease that can act on a wide variety of RNA- and DNA-containing substrates and exhibits preference for degrading RNA in model Okazaki fragments. Together, our data showed that RNase HIII and DNA polymerase I provide the primary pathway for Okazaki fragment maturation, whereas YpcP also contributes to the removal of RNA from an Okazaki fragmentin vitro.IMPORTANCEAll cells are required to resolve the different types of RNA-DNA hybrids that formin vivo. When RNA-DNA hybrids persist, cells experience an increase in mutation rate and problems with DNA replication. Okazaki fragment synthesis on the lagging strand requires an RNA primer to begin synthesis of each fragment. The mechanism of RNA removal from Okazaki fragments remains unknown in bacteria that lack RNase HI. We examined Okazaki fragment processingin vitroand found that RNase HIII in conjunction with DNA polymerase I represent the most efficient repair pathway. We also assessed the contribution of YpcP and found that YpcP is a 5′ to 3′ exonuclease that prefers RNA substrates with activity on Okazaki and flap substratesin vitro.

scholarly journals Flexibility of Eukaryotic Okazaki Fragment Maturation through Regulated Strand Displacement Synthesis

2008 ◽  
Vol 283 (49) ◽  
pp. 34129-34140 ◽  
Author(s):  
Carrie M. Stith ◽  
Joan Sterling ◽  
Michael A. Resnick ◽  
Dmitry A. Gordenin ◽  
Peter M. Burgers
Keyword(s):  
Strand Displacement ◽  
Okazaki Fragment ◽  
Okazaki Fragment Maturation

scholarly journals Supramolecular Structures of the Dictyostelium Lamin NE81

Cells ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 162
Author(s):  
Marianne Grafe ◽  
Petros Batsios ◽  
Irene Meyer ◽  
Daria Lisin ◽  
Otto Baumann ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Nuclear Envelope ◽  
Model Organism ◽  
Super Resolution ◽  
Nuclear Lamina ◽  
Stimulated Emission ◽  
Structural Level ◽  
Animal Cells ◽  
Nuclear Lamins

Nuclear lamins are nucleus-specific intermediate filaments (IF) found at the inner nuclear membrane (INM) of the nuclear envelope (NE). Together with nuclear envelope transmembrane proteins, they form the nuclear lamina and are crucial for gene regulation and mechanical robustness of the nucleus and the whole cell. Recently, we characterized Dictyostelium NE81 as an evolutionarily conserved lamin-like protein, both on the sequence and functional level. Here, we show on the structural level that the Dictyostelium NE81 is also capable of assembling into filaments, just as metazoan lamin filament assemblies. Using field-emission scanning electron microscopy, we show that NE81 expressed in Xenopous oocytes forms filamentous structures with an overall appearance highly reminiscent of Xenopus lamin B2. The in vitro assembly properties of recombinant His-tagged NE81 purified from Dictyostelium extracts are very similar to those of metazoan lamins. Super-resolution stimulated emission depletion (STED) and expansion microscopy (ExM), as well as transmission electron microscopy of negatively stained purified NE81, demonstrated its capability of forming filamentous structures under low-ionic-strength conditions. These results recommend Dictyostelium as a non-mammalian model organism with a well-characterized nuclear envelope involving all relevant protein components known in animal cells.

scholarly journals Creation and Removal of Embedded Ribonucleotides in Chromosomal DNA during Mammalian Okazaki Fragment Processing

1997 ◽  
Vol 272 (36) ◽  
pp. 22591-22599 ◽  
Author(s):  
Jeffrey A. Rumbaugh ◽  
Richard S. Murante ◽  
Shuying Shi ◽  
Robert A. Bambara
Keyword(s):  
Chromosomal Dna ◽  
Okazaki Fragment ◽  
Okazaki Fragment Processing

scholarly journals Involvement of Vts1, a structure-specific RNA-binding protein, in Okazaki fragment processing in yeast

2009 ◽  
Vol 38 (5) ◽  
pp. 1583-1595 ◽  
Author(s):  
Chul-Hwan Lee ◽  
Yong-Keol Shin ◽  
Thi Thu Huong Phung ◽  
Jae Seok Bae ◽  
Young-Hoon Kang ◽  
...  
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Okazaki Fragment ◽  
Okazaki Fragment Processing

Electron microscopy studies of Type III CRISPR machines in Sulfolobus solfataricus

2013 ◽  
Vol 41 (6) ◽  
pp. 1427-1430 ◽  
Author(s):  
Giuseppe Cannone ◽  
Mariam Webber-Birungi ◽  
Laura Spagnolo
Keyword(s):  
Electron Microscopy ◽  
Immune System ◽  
Unit Length ◽  
Sulfolobus Solfataricus ◽  
Adaptive Immune System ◽  
Particle Analysis ◽  
Single Particle Analysis ◽  
Type Iii Effector ◽  
Type Iii ◽  
Adaptive Immune

The CRISPR (clustered regularly interspaced short palindromic repeats) system is an adaptive immune system that targets viruses and other mobile genetic elements in bacteria and archaea. Cells store information of past infections in their genome in repeat–spacer arrays. After transcription, these arrays are processed into unit-length crRNA (CRISPR RNA) that is loaded into effector complexes encoded by Cas (CRISPR-associated) genes. CRISPR–Cas complexes target invading nucleic acid for degradation. CRISPR effector complexes have been classified into three main types (I–III). Type III effector complexes share the Cas10 subunit. In the present paper, we discuss the structures of the two Type III effector complexes from Sulfolobus solfataricus, SsoCSM (subtype III-A) and SsoCMR (subtype III-B), obtained by electron microscopy and single particle analysis. We also compare these structures with Cascade (CRISPR-associated complex for antiviral defence) and with the RecA nucleoprotein.

Effect of polyamine deficiency on proteins involved in Okazaki fragment maturation

2008 ◽  
Vol 32 (12) ◽  
pp. 1467-1477 ◽  
Author(s):  
Veronica M. Johansson ◽  
Maria Falck Miniotis ◽  
Cecilia Hegardt ◽  
Göran Jönsson ◽  
Johan Staaf ◽  
...  
Keyword(s):  
Okazaki Fragment ◽  
Okazaki Fragment Maturation

scholarly journals Okazaki Fragment Processing-independent Role for Human Dna2 Enzyme during DNA Replication

2012 ◽  
Vol 287 (26) ◽  
pp. 21980-21991 ◽  
Author(s):  
Julien P. Duxin ◽  
Hayley R. Moore ◽  
Julia Sidorova ◽  
Kenneth Karanja ◽  
Yuchi Honaker ◽  
...  
Keyword(s):  
Dna Replication ◽  
Okazaki Fragment ◽  
Okazaki Fragment Processing
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