Multiple physiological roles of both specific and non-specific DNA binding modes of HU protein in Escherichia coli
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ABSTRACT: The histone-like protein HU is crucial for genome organization and the expression of many genes in Escherichia coli. HU binds DNA independent of any specific nucleotide sequence but exhibits two binding affinities; low-affinity to any B-DNA (non-specific) and high affinity to DNA with distortions like kinks and cruciforms (structure-specific). We validated and defined the three conserved lysine residues, K3, K18, and K83, in HU as critical amino acid residues for the non-specific binding and the conserved P63 together with the lysine residues critical for the structure-specific binding both in vitro and in vivo. By determining the effects of disrupting the two DNA binding modes on various HU-dependent physiological processes, we demonstrate that the DNA structure-specific binding regulates expression of many genes including those involved in DNA metabolic processes that control chromosome maintenance and partition. On the other hand, HU associates with the chromosome at numerous sites primarily through the lysines-mediated non-specific binding and control chromosome dynamics and structural maintenance. Thus, we demonstrate that two different DNA binding mode of HU plays separate roles: (i) The high-affinity DNA structure-specific binding regulates many distinct DNA metabolic processes, primarily through transcription regulation. (ii) Its low-affinity, non-specific binding directly helps general organization of the genome.
Project description:Lysine acetylation is a prevalent post-translational modification in both eukaryotes and prokaryotes. Whereas this modification is known to play pivotal roles in eukaryotes, the function and extent of this modification in prokaryotic cells remain largely unexplored. Here we report the acetylome of a pair of antibiotic-sensitive and -resistant nosocomial pathogen Acinetobacter baumannii SK17-S and SK17-R. A total of 145 lysine acetylation sites on 125 proteins was identified, and there are 23 acetylated proteins found in both strains, including histone-like protein HU which was found to be acetylated at Lys13. HU is a dimeric DNA-binding protein critical for maintaining chromosomal architecture and other DNA-dependent functions. To analyze the effects of site-specific acetylation, homogenously Lys13-acetylated HU protein, HU(K13ac) was prepared by genetic code expansion. Whilst not exerting an obvious effect on the oligomeric state, Lys13 acetylation alters both the thermal stability and DNA binding kinetics of HU. Accordingly, this modification likely destabilizes the chromosome structure and regulates bacterial gene transcription. By preparing and characterizing site-specifically acetylated bacterial protein for the first time, this work indicates that acetyllysine plays an important role in bacterial epigenetics and presents a promising target against bacterial infections.
Project description:HU is a conserved prokaryotic nucleoid-associated protein known for its role in DNA binding and maintaining negative supercoils in DNA. HU also binds to a few RNAs but the natural targets are unknown. To understand the biology of HU interactions with RNA in Escherichia coli, potential HU-RNA in vivo complexes were immunoprecipitated and bound RNAs were examined by hybridization to whole-genome tiling arrays. We observed HU associations with rRNA, multiple tRNAs, a few sRNAs and a small, distinct set of mRNAs. More importantly, we also identified associations between HU and ten intragenic non-coding (nonc) RNAs, two of which are homologous to the annotated Bacterial Interspersed Mosaic Elements (BIME) and boxC elements; the other eight RNAs have not been annotated. We confirmed binding of HU to BIME RNA in vitro. Since HU and some unidentified RNAs have been implicated in the nucleoid structure, we explored an idea that HU, along with some or all of the nonc RNA species discovered here bind to BIME and/or boxC repeats to condense chromosomal DNA in a specific fashion. We propose that nonc RNAs may interact with both HU and DNA repeats to form ternary complexes, which are the anchoring points separating different chromosomal domains.
Project description:HupA and HupS are nucleoid associated proteins, homologic to E. coli HU protein. Their binding to DNA changes chromosome structure, protects DNA from damage and influences gene expression. The goal of RNA-seq experiment was to determine HupA and HupS regulons during growth in liquid medium. Dataset contains four strains (hupA, hupS, hupAhupS deletion mutants and wild type), two time points (exponential and stationary growth) and two growth conditions (standard media and osmotic stress).
Project description:We previously demonstrated that inactivation of the replication checkpoint via a mec1 mutation led to chromosome breakage at replication forks initiated from virtually all origins of replication, after transient exposure to hydroxyurea (HU), an inhibitor of ribonucleotide reductase. Furthermore, we have shown that chromosomes break at replication forks that have suffered single-stranded DNA (ssDNA) formation. Here we sought to determine whether all replication forks containing ssDNA gaps have equal probability of producing double strand breaks (DSBs) when cells attempt to recover from HU exposure. We devised a new methodology, Break-Seq, that combines our previously described DSB labeling with NextGen sequencing to map chromosome breaks with improved sensitivity and resolution. We show that DSBs preferentially occur at genes transcriptionally induced by HU. Notably, different subsets of the HU-induced genes produced DSBs in MEC1 and mec1 cells as replication forks traversed greater distance in MEC1 cells than in mec1 cells during the recovery from HU. Specifically, while MEC1 cells exhibited chromosome breakage at stress-response transcription factors, mec1 cells predominantly suffered chromosome breakage at transporter genes, many of which are the substrates of the said transcription factors. We propose that HU-induced chromosome fragility arises at higher frequency near HU-induced genes as a result of destabilized replication forks encountering transcription factor binding and/or the act of transcription. Our model provides an explanation for a long-standing problem in chromosome biology: why different replication inhibitors produce different spectra of chromosome breakage? We propose that different inhibitors elicit different transcription responses as well as destabilize replication forks, and, when the two processes collide, ssDNA at the replication fork suffers further strand breakage, causing DSBs.
Project description:We previously demonstrated that inactivation of the replication checkpoint via a mec1 mutation led to chromosome breakage at replication forks initiated from virtually all origins of replication, after transient exposure to hydroxyurea (HU), an inhibitor of ribonucleotide reductase. Furthermore, we have shown that chromosomes break at replication forks that have suffered single-stranded DNA (ssDNA) formation. Here we sought to determine whether all replication forks containing ssDNA gaps have equal probability of producing double strand breaks (DSBs) when cells attempt to recover from HU exposure. We devised a new methodology, Break-Seq, that combines our previously described DSB labeling with NextGen sequencing to map chromosome breaks with improved sensitivity and resolution. We show that DSBs preferentially occur at genes transcriptionally induced by HU. Notably, different subsets of the HU-induced genes produced DSBs in MEC1 and mec1 cells as replication forks traversed greater distance in MEC1 cells than in mec1 cells during the recovery from HU. Specifically, while MEC1 cells exhibited chromosome breakage at stress-response transcription factors, mec1 cells predominantly suffered chromosome breakage at transporter genes, many of which are the substrates of the said transcription factors. We propose that HU-induced chromosome fragility arises at higher frequency near HU-induced genes as a result of destabilized replication forks encountering transcription factor binding and/or the act of transcription. Our model provides an explanation for a long-standing problem in chromosome biology: why different replication inhibitors produce different spectra of chromosome breakage? We propose that different inhibitors elicit different transcription responses as well as destabilize replication forks, and, when the two processes collide, ssDNA at the replication fork suffers further strand breakage, causing DSBs.
Project description:IHF and HU are two heterodimeric nucleoid associated proteins (NAP) that belong to the same protein family but interact differently with the DNA. IHF is a sequence-specific DNA-binding protein that bends the DNA by over 160o. HU is the most conserved NAP which binds non-specifically to duplex DNA with a particular preference for targeting nicked and bent DNA. Despite their importance, the in-vivo interactions of the two proteins to the DNA remain to be described at a high resolution and on a genome-wide scale. Further, the effects of these proteins on gene expression on a global scale remain contentious. Finally, the contrast between the functions of the homo- and heterodimeric forms of these proteins deserves the attention of further study. Here we present a genome-scale study of HU- and IHF-binding to the E. coli K12 chromosome using ChIP-seq. We also perform microarray analysis of gene expression in single- and double-deletion mutants of each protein to identify their regulons. This data is for microarrays measuring gene expression changes in the various ihf mutants when compared to the wildtype.<br>
Project description:Mammalian transcription factors (TFs) differ broadly in their nuclear mobility and sequence-specific/ non-specific DNA binding affinity. How these properties affect the ability of TFs to occupy their specific binding sites in the genome and modify the epigenetic landscape is unclear. Here we combined live cell quantitative measurements of mitotic chromosome binding (MCB) of 502 TFs, measurements of TF mobility by fluorescence recovery after photobleaching, single molecule imaging of DNA binding in live cells, and genome-wide mapping of TF binding and chromatin accessibility. MCB scaled with interphase properties such as association with DNA-rich compartments, mobility, as well as large differences in genome-wide specific site occupancy that correlated with TF impact on chromatin accessibility. As MCB is largely mediated by electrostatic, non-specific TF-DNA interactions, our data suggests that non-specific DNA binding of TFs enhances their search for specific sites and thereby their impact on the accessible chromatin landscape.
Project description:Mammalian transcription factors (TFs) differ broadly in their nuclear mobility and sequence-specific/ non-specific DNA binding affinity. How these properties affect the ability of TFs to occupy their specific binding sites in the genome and modify the epigenetic landscape is unclear. Here we combined live cell quantitative measurements of mitotic chromosome binding (MCB) of 502 TFs, measurements of TF mobility by fluorescence recovery after photobleaching, single molecule imaging of DNA binding in live cells, and genome-wide mapping of TF binding and chromatin accessibility. MCB scaled with interphase properties such as association with DNA-rich compartments, mobility, as well as large differences in genome-wide specific site occupancy that correlated with TF impact on chromatin accessibility. As MCB is largely mediated by electrostatic, non-specific TF-DNA interactions, our data suggests that non-specific DNA binding of TFs enhances their search for specific sites and thereby their impact on the accessible chromatin landscape.
Project description:We previously demonstrated that inactivation of the replication checkpoint via a mec1 mutation led to chromosome breakage at replication forks initiated from virtually all origins of replication, after transient exposure to hydroxyurea (HU), an inhibitor of ribonucleotide reductase. Furthermore, we have shown that chromosomes break at replication forks that have suffered single-stranded DNA (ssDNA) formation. Here we sought to determine whether all replication forks containing ssDNA gaps have equal probability of producing double strand breaks (DSBs) when cells attempt to recover from HU exposure. We devised a new methodology, Break-Seq, that combines our previously described DSB labeling with NextGen sequencing to map chromosome breaks with improved sensitivity and resolution. We show that DSBs preferentially occur at genes transcriptionally induced by HU. Notably, different subsets of the HU-induced genes produced DSBs in MEC1 and mec1 cells as replication forks traversed greater distance in MEC1 cells than in mec1 cells during the recovery from HU. Specifically, while MEC1 cells exhibited chromosome breakage at stress-response transcription factors, mec1 cells predominantly suffered chromosome breakage at transporter genes, many of which are the substrates of the said transcription factors. We propose that HU-induced chromosome fragility arises at higher frequency near HU-induced genes as a result of destabilized replication forks encountering transcription factor binding and/or the act of transcription. Our model provides an explanation for a long-standing problem in chromosome biology: why different replication inhibitors produce different spectra of chromosome breakage? We propose that different inhibitors elicit different transcription responses as well as destabilize replication forks, and, when the two processes collide, ssDNA at the replication fork suffers further strand breakage, causing DSBs. We queried the yeast genome for gene expression after cells were treated with 200 mM hydroxyurea during S phase. Samples were collected from 1) cells synchronized in G1 phase by alpha factor; 2) cells released from G1 into medium containing 200 mM hydroxyurea for 1 h; 3) cells recovering in fresh medium without hydroxyurea for 30 and 60 min after the 1 h exposure to HU. These samples are referred to as G1, HU 1h, R30, and R60, respectively. The strains from which the samples were collected are indicated before the time point, e.g. mec1_G1 or MEC1_R30. Stranded mRNA libraries were prepared according to manufacturer's suggestion and sequenced on Illumina MiSeq with paired-end reads of 75 bp.