The N-Terminal Region Is Important for the Nuclease Activity and Thermostability of the Flap Endonuclease-1 from<i>Sulfolobus tokodaii</i>

Horie, Masanori; Fukui, Kôichi; Xie, Minjue; Kageyama, Yuji; Hamada, Kazuo; Sakihama, Yuri; Sugimori, Kenji; Matsumoto, Kazuko

doi:10.1271/bbb.60326

Cited by 7 publications

(5 citation statements)

References 29 publications

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“…The recombinant proteins were purified on a HiTrap S column (5 ml; GE) preequilibrated with buffer B, and the column was washed with buffer C (20 mM Tris-Cl [pH 6.8], 1 M KCl, 1 mM EDTA, 1 mM DTT, and 10% [wt/vol] glycerol). S. solfataricus chromatin and replication proteins RFC, Pri1/Pri2, Dpo4, PCNA, Topo III, PolB1, GINS, FEN1, Cren7, and Sso7d2 were overproduced in E. coli and purified as described previously (8,12,15,17,25,26,35,39,43,53).…”

Section: Purification and Identification Of Akmt From S Islandicus mentioning

confidence: 99%

Identification and Characterization of a Highly Conserved Crenarchaeal Protein Lysine Methyltransferase with Broad Substrate Specificity

et al. 2012

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Protein lysine methylation occurs extensively in the Crenarchaeota, a major kingdom in the Archaea. However, the enzymes responsible for this type of posttranslational modification have not been found. Here we report the identification and characterization of the first crenarchaeal protein lysine methyltransferase, designated aKMT, from the hyperthermophilic crenarchaeon Sulfolobus islandicus. The enzyme was capable of transferring methyl groups to selected lysine residues in a substrate protein using S-adenosyl-L-methionine (SAM) as the methyl donor. aKMT, a non-SET domain protein, is highly conserved among crenarchaea, and distantly related homologs also exist in Bacteria and Eukarya. aKMT was active over a wide range of temperatures, from ϳ25 to 90°C, with an optimal temperature at ϳ60 to 70°C. Amino acid residues Y9 and T12 at the N terminus appear to be the key residues in the putative active site of aKMT, as indicated by sequence conservation and site-directed mutagenesis. Although aKMT was identified based on its methylating activity on Cren7, the crenarchaeal chromatin protein, it exhibited broad substrate specificity and was capable of methylating a number of recombinant Sulfolobus proteins overproduced in Escherichia coli. The finding of aKMT will help elucidate mechanisms underlining extensive protein lysine methylation and the functional significance of posttranslational protein methylation in crenarchaea.

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Section: Purification and Identification Of Akmt From S Islandicus mentioning

confidence: 99%

Identification and Characterization of a Highly Conserved Crenarchaeal Protein Lysine Methyltransferase with Broad Substrate Specificity

et al. 2012

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“…Moreover, the protein sequence itself can be either truncated or extended, leading to errors in bioinformatics protein characterization (function, localization, etc.) and, obviously, to major difficulties in protein expression experiments (Trivedi et al 2004;Horie et al 2007). The second highly prejudicial error encountered in prokaryotic genome annotation is under-prediction of small genes or genes exhibiting an unusual composition.…”

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confidence: 99%

Ortho-proteogenomics: Multiple proteomes investigation through orthology and a new MS-based protocol

Gallien¹,

Perrodou²,

Carapito³

et al. 2008

Genome Res.

102

115

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The progress in sequencing technologies irrigates biology with an ever-increasing number of genome sequences. In most cases, the gene repertoire is predicted in silico and conceptually translated into proteins. As recently highlighted, the predicted genes exhibit frequent errors, particularly in start codons, with a serious impact on subsequent biological studies. A new "ortho-proteogenomic" approach is presented here for the annotation refinement of multiple genomes at once. It combines comparative genomics with an original proteomic protocol that allows the characterization of both N-terminal and internal peptides in a single experiment. This strategy was applied to the Mycobacterium genus with Mycobacterium smegmatis as the reference, and identified 946 distinct proteins, including 443 characterized N termini. These experimental data allowed the correction of 19% of the characterized start codons, the identification of 29 proteins missed during the annotation process, and the curation, thanks to comparative genomics, of 4328 sequences of 16 other Mycobacterium proteomes.

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“…Even precise removal of the lesion from the annealed oligonucleotide by an endonuclease was expected to negate transformation, because a 1-nt, 59 flap on the 39 side of the repair gap provides a favorable substrate for removal by the Sulfolobus Fen1/XPG homolog (Horie et al 2007;Hutton et al 2008) but not for the highly selective Sulfolobus ligase (Lai et al 2002). We evaluated the feasibility of OMT as a probe of lesion bypass in Sulfolobus experimentally by placing a chemically stabilized abasic site (THF spacer) in three transforming oligonucleotides.…”

Section: Molecular Assays Of Tls In the Sulfolobus Chromosomementioning

confidence: 99%

Lesion-Induced Mutation in the Hyperthermophilic Archaeon Sulfolobus acidocaldarius and Its Avoidance by the Y-Family DNA Polymerase Dbh

Sakofsky

Grogan

2015

Genetics

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Hyperthermophilic archaea offer certain advantages as models of genome replication, and Sulfolobus Y-family polymerases Dpo4 (S. solfataricus) and Dbh (S. acidocaldarius) have been studied intensively in vitro as biochemical and structural models of trans-lesion DNA synthesis (TLS). However, the genetic functions of these enzymes have not been determined in the native context of living cells. We developed the first quantitative genetic assays of replication past defined DNA lesions and error-prone motifs in Sulfolobus chromosomes and used them to measure the efficiency and accuracy of bypass in normal and dbh 2 strains of Sulfolobus acidocaldarius. Oligonucleotide-mediated transformation allowed low levels of abasic-site bypass to be observed in S. acidocaldarius and demonstrated that the local sequence context affected bypass specificity; in addition, most erroneous TLS did not require Dbh function. Applying the technique to another common lesion, 7,8-dihydro-8-oxo-deoxyguanosine (8-oxo-dG), revealed an antimutagenic role of Dbh. The efficiency and accuracy of replication past 8-oxo-dG was higher in the presence of Dbh, and up to 90% of the Dbh-dependent events inserted dC. A third set of assays, based on phenotypic reversion, showed no effect of Dbh function on spontaneous 21 frameshifts in mononucleotide tracts in vivo, despite the extremely frequent slippage at these motifs documented in vitro. Taken together, the results indicate that a primary genetic role of Dbh is to avoid mutations at 8-oxo-dG that occur when other Sulfolobus enzymes replicate past this lesion. The genetic evidence that Dbh is recruited to 8-oxo-dG raises questions regarding the mechanism of recruitment, since Sulfolobus spp. have eukaryotic-like replisomes but no ubiquitin.KEYWORDS trans-lesion DNA synthesis (TLS); abasic site; 7,8-dihydro-8-oxo-deoxyguanosine; oligonucleotide-mediated transformation T HE fact that DNA damage occurs in all living cells poses a threat to the accurate replication and partitioning of their genomes. Cells counter this threat with an array of diverse damage-coping systems, some of which repair the DNA, whereas others enable replication to continue past persistent lesions. Lesion bypass, also termed "damage tolerance," can follow various alternatives, which are broadly distinguished as error-free vs. error-prone (Lehmann et al. 2007). The error-free pathways generally use recombinationassociated functions to pair a strand, newly synthesized on intact template, to the damaged DNA strand; the mechanisms for this include transient reversal of the replication fork and strand exchange at gaps left behind the fork (Sale 2012). These mechanisms bypass lesions accurately, although they can promote other forms of genetic instability, such as ectopic recombination between dispersed repeats (Izhar et al. 2013).Error-prone mechanisms of lesion bypass, in contrast, use specialized, trans-lesion synthesis (TLS) polymerases to continue strand elongation using the damaged template; most of these enzymes belong to t...

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The N-Terminal Region Is Important for the Nuclease Activity and Thermostability of the Flap Endonuclease-1 fromSulfolobus tokodaii

Cited by 7 publications

References 29 publications

Identification and Characterization of a Highly Conserved Crenarchaeal Protein Lysine Methyltransferase with Broad Substrate Specificity

Identification and Characterization of a Highly Conserved Crenarchaeal Protein Lysine Methyltransferase with Broad Substrate Specificity

Ortho-proteogenomics: Multiple proteomes investigation through orthology and a new MS-based protocol

Lesion-Induced Mutation in the Hyperthermophilic Archaeon Sulfolobus acidocaldarius and Its Avoidance by the Y-Family DNA Polymerase Dbh

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