Abstract
AbstractBackgroundHigh mobility group box (HMGB) family protein Ixr1 has been shown to be involved in DNA damage repair, however, its role and mechanism remain largely unclear.MethodsGenes ofS. cerevisiaewere deleted or tagged with myc, GFP, or mcherry using the lithium acetate method. Sensitivity of strains to hydroxyurea (HU), methyl methanesulfonate (MMS), camptothe-cin (CPT), 4-nitroquinoline N-oxide (4-NQ), or Zeocin was tested. Distribution of GFP or mcherry fusion proteins was visualized with laser scanning confocal microscopy. RNA-seq was used to determine differential gene expression between mutant and control strains.ResultsIxr1 deletion (ixr1Δ) mutant strain was sensitive to HU. Additionally, phosphorylation of effector of DNA damage checkpoint kinase Rad53 was lower in ixr1Δ than WT. Deletion of DNA damage checkpoint mediators ixr1Δ Rad9Δ was more sensitive to HU than ixr1Δ or Rad9Δ, and ixr1Δ mrc1Δ had similar sensitivity to HU as mrc1Δ but stronger than ixr1Δ. Deletion of ribonucleotide reductase inhibitors sml1Δ or crt10Δ didn’t reduce the sensitivity of ixr1Δ induced by HU. Repli-cation fork nuclease exo1Δ ixr1Δ or helicase sgs1Δ ixr1Δ double deletions were more sensitive to HU than single deletion. In addition, laser scanning confocal microscopy imaging indicated that in response to HU, Ixr1 may be in the same pathway as Mrc1, possibly downstream. Gene Ontol-ogy enrichment analysis of differentially expressed genes (DEGs) between ixr1Δ and wildtype, untreated and treated with HU, confirmed that Ixr1 plays an important role in regulating the transcription of genes related to DNA replication or DNA damage repair. We also found that, re-gardless of HU exposure, Ixr1 localized to the nucleus and may bind DNA through its two HMG-boxes.ConclusionIxr1 participates in the DNA replication stress response through a DNA damage checkpoint pathway mediated by Mrc1, and regulates expression of genes related to DNA damage repair.
Publisher
Cold Spring Harbor Laboratory