Erstand how increased dNTP levels in sml1 and rfx1 (deletion) mutantsErstand how increased dNTP levels

Erstand how increased dNTP levels in sml1 and rfx1 (deletion) mutants
Erstand how increased dNTP levels in sml1 and rfx1 (deletion) Chaetocin msds mutants relate to increased Ty1 mobility, we investigated multiple steps within the Ty lifecycle. We quantified transposition and cDNA levels in wild type, rfx1 and sml1 strains at varying temperatures. Southern blot analysis demonstrated that cDNA levels did not differ between the wild type and mutant strains as temperature increases, indicating thatO’Donnell et al. Mobile DNA 2010, 1:23 http://www.mobilednajournal.com/content/1/1/Page 3 ofDNA damage/stalled replication forkMrc1:replication pausesNTPs Rad9:replication blocksdNTPs active RNR PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/26437915 ti enzymesensor complex p inactive Sml1 Rad53 DunP active Sml1 inactive RNR enzymeP inactive Rfx1 active Rfx1 (repressor)translationRNR genes (RNR2-RNR4)Figure 1 Schematic of the ribonucleotide reductase (RNR) induction pathway. DNA damage or stalled replication forks during S-phase activates a sensor complex PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27488460 that recruits Mec1 to the site. Mec1 then activates a mediator kinase; either Mrc1 at stalled replication forks, or Rad9 at damaged sites. The activated mediator kinase then activates the effector kinase Rad53, which subsequently phosphorylates Dun1. The activated Dun1 protein phosphorylates the transcriptional repressor Rfx1 (Crt1), thereby inactivating it and allowing for increased transcription of the RNR genes. Sml1 binds and keeps inactive the large RNR1 subunit; Dun1 phosphorylation of Sml1 causes it to release RNR1, and the enzyme becomes active. RNR converts NTPs to dNTPs as needed for DNA synthesis and repair. Only the pathway components most relevant to this study are shown.increased Ty1 mobility is not a result of increased cDNA synthesis in the deletion strains. Homologous recombination efficiency was increased in both rfx1 and sml1 strains at high temperatures, suggesting that the increased mobility results from increased homologous recombination of Ty1 cDNA. Mobility assays were also conducted in the presence of the dNTP reducing agent, hydroxyurea (HU), which eliminated the high temperature phenotype in both rfx1 and sml1 strains. Our results show that dNTP concentrations are a limiting resource for Ty1 mobility at high temperatures.ResultsGenetic screen for regulators of high temperature transpositionTy1 transposition is temperature sensitive, mainly due to inactivity of the protease enzyme and reduction of protein levels at high temperatures [3]. To study specific host regulation of transposition, we screened a yeast deletion library for mutants that transpose under hightemperature conditions of 34 [34]. An aliquot of the MATa deletion pool was transformed with a galactoseinducible Ty1 element (pGTy1) marked with his3AI on plasmid pGTy1H3mhis3AI [35]. With this plasmid, transposition is assayed by plating cells onto a medium containing galactose to induce transposition, followed by plating to a medium lacking histidine to select for Ty1 mobility. Upon splicing and subsequent reverse transcription of the his3AI marker gene, a functional HIS3 gene sequence can be incorporated into the yeast genome, either by integration via the Ty1 integrase or by recombination by the host cellular machinery. The resulting His-positive papillae are collectively referred to as pGTy1 mobility events, because this assay cannot distinguish between integration and recombination. pGTy1H3mhis3AI transformants of a MATa deletion pool were induced on galactose medium at 34 and subsequently screened for growth on synthetic complete medium lack.