Degradation of Escherichia coli RecN Aggregates by ClpXP Protease and Its Implications for DNA Damage Tolerance

Abstract

Protein degradation in bacteria plays a dynamic and critical role in the cellular response to environmental stimuli such as heat shock and DNA damage and in removing damaged proteins or protein aggregates. Escherichia coli recN is a member of the structural maintenance of chromosomes family and is required for DNA double strand break (DSB) repair. This study shows that RecN protein has a short half-life and its degradation is dependent on the cytoplasmic protease ClpXP and a degradation signal at the C terminus of RecN. In cells with DNA DSBs, green fluorescent protein-RecN localized in discrete foci on nucleoids and formed visible aggregates in the cytoplasm, both of which disappeared rapidly in wild-type cells when DSBs were repaired. In contrast, in ΔclpX cells, RecN aggregates persisted in the cytoplasm after release from DNA damage. Furthermore, analysis of cells experiencing chronic DNA damage revealed that proteolytic removal of RecN aggregates by ClpXP was important for cell viability. These data demonstrate that ClpXP is a critical factor in the cellular clearance of cytoplasmic RecN aggregates from the cell and therefore plays an important role in DNA damage tolerance. Protein degradation in bacteria plays a dynamic and critical role in the cellular response to environmental stimuli such as heat shock and DNA damage and in removing damaged proteins or protein aggregates. Escherichia coli recN is a member of the structural maintenance of chromosomes family and is required for DNA double strand break (DSB) repair. This study shows that RecN protein has a short half-life and its degradation is dependent on the cytoplasmic protease ClpXP and a degradation signal at the C terminus of RecN. In cells with DNA DSBs, green fluorescent protein-RecN localized in discrete foci on nucleoids and formed visible aggregates in the cytoplasm, both of which disappeared rapidly in wild-type cells when DSBs were repaired. In contrast, in ΔclpX cells, RecN aggregates persisted in the cytoplasm after release from DNA damage. Furthermore, analysis of cells experiencing chronic DNA damage revealed that proteolytic removal of RecN aggregates by ClpXP was important for cell viability. These data demonstrate that ClpXP is a critical factor in the cellular clearance of cytoplasmic RecN aggregates from the cell and therefore plays an important role in DNA damage tolerance. DNA double strand breaks (DSBs) 2The abbreviations used are: DSB, DNA double strand break; SMC, structural maintenance of chromosome; GFP, green fluorescent protein; LB, Luria Bertani; DAPI, 4′,6-diamidino-2-phenylindole; MMC, mitomycin C. 2The abbreviations used are: DSB, DNA double strand break; SMC, structural maintenance of chromosome; GFP, green fluorescent protein; LB, Luria Bertani; DAPI, 4′,6-diamidino-2-phenylindole; MMC, mitomycin C. are major threats to the genomic integrity of cells. DSBs can be caused by exogenous and endogenous agents such as ionizing radiation and chemical mutagens and by endogenously produced radicals or as a result of replication arrest and/or collapse of the replication fork (1Cox M.M. Goodman M.F. Kreuzer K.N. Sherratt D.J. Sandler S.J. Marians K.J. Nature. 2000; 404: 37-41Crossref PubMed Scopus (861) Google Scholar, 2Kowalczykowski S.C. Trends Biochem. Sci. 2000; 25: 156-165Abstract Full Text Full Text PDF PubMed Scopus (562) Google DSBs are and result in such as genomic and the of DSBs is for cell and for the integrity of the Escherichia the of is for the of DNA by its S.C. Trends Biochem. Sci. 2000; 25: 156-165Abstract Full Text Full Text PDF PubMed Scopus (562) Google These DNA DNA a the which is can when the is by coli recN has in the of recN that on recN in response to C. PubMed Scopus Google Scholar, PubMed Google of recN in C. PubMed Scopus Google Scholar, PubMed Scopus Google that RecN is of the of in to the of the in recN to PubMed Scopus Google and is that the RecN protein is required for of DSBs PubMed Scopus Google Scholar, PubMed Google was that RecN is required to breaks at a by PubMed Scopus Google Furthermore, recN are to ionizing radiation and mitomycin C to PubMed Scopus Google RecN of be in the of DNA DSBs by an and and and which are of the maintenance of family Full Text Full Text PDF PubMed Scopus Google Scholar, Sci. PubMed Scopus Google proteins in DNA replication and/or by and in and cells. that proteins in cell and DNA and that the of proteins and are by PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, DNA PubMed Scopus Google Scholar, 25: PubMed Scopus Google that RecN protein is by the cytoplasmic protease ClpXP in a dependent the signal at the C terminus of RecN. Furthermore, that in ΔclpX cells, DNA RecN formed visible aggregates in the cytoplasm that persisted after release from DNA damage and were to cell These data demonstrate that proteolytic removal of RecN aggregates plays an important role in after release from DNA damage and is therefore for cell and for coli and DNA were as by and for Escherichia coli and and and were used of ionizing radiation was with an of and that used in study were with Sci. 2000; PubMed Scopus Google for and were by PubMed Scopus Google was a from PubMed Scopus Google the and of recN was the PubMed Scopus Google RecN was with an green fluorescent protein at its recN or was by the recN or PubMed Scopus Google recN were by and with the wild-type recN of of was by DNA to C cells were to in Luria at was and were at the was by of the on or were after are the of at and degradation were as PubMed Scopus Google were with for at of was to protein were at the and were rapidly in in at and and by were by as PubMed Scopus Google was with the RecN was were with and with of analysis were by was on a PubMed Scopus Google of or are in the in the and of RecN in and cells. were to to DNA DSBs, and RecN was In wild-type cells to ionizing RecN protein was which is with the that coli recN is a member of the C. 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This is by the that coli RecN protein protein aggregates PubMed Scopus Google and protein aggregates are to at cell in the cytoplasm in that the of recN is and in cells with DNA damage. of RecN foci in was in cells from the were to to and to to cells were and/or to that cytoplasmic of foci formed in cells to both cytoplasmic and foci formed in cells to and foci were in were in cells with DNA damage These that DNA DSBs of RecN after from foci were in ΔclpX cells. and cells were with for to MMC, and to for at with for cells were and of cells foci at analysis revealed that is after to in wild-type cells, in ΔclpX cells in wild-type cells, and cytoplasmic foci disappeared rapidly and In contrast, in ΔclpX cells, foci disappeared after to MMC, cytoplasmic foci persisted for and were to cells when cell in and Furthermore, persisted in the of cytoplasmic RecN aggregates after release from DNA damage These data that ClpXP cytoplasmic RecN foci in cells with DNA cytoplasmic RecN aggregates after release from cells were with for MMC, and at the after release from cells were by fluorescent the of cell were by with analysis of after release from and the of cells with RecN foci and cytoplasmic RecN of the cells were of after release from DNA damage. or were as in shows and of cells after removal of of RecN by ClpXP for that to protein aggregates can and Nature. PubMed Scopus Google Scholar, PubMed Scopus Google the role of RecN aggregates in and/or DNA in cells with DNA damage was by cell in and ΔclpX cells with cells were with for cell was in cells in wild-type cells, in ΔclpX and wild-type cells when cells were with for of cell was in ΔclpX cells in wild-type cells of cytoplasmic RecN aggregates are for cell the of cells a that RecN or cell of cells was to the of wild-type RecN after of to was the of wild-type RecN after of to These that cytoplasmic RecN aggregates cell in cells with chronic DNA the of the of RecN for degradation by for is that the RecN aggregates in ΔclpX or cells to of RecN protein and/or DNA proteins and with DNA caused by RecN aggregates is to cells with DNA wild-type and ΔclpX cells in the of DNA damage RecN aggregates were in wild-type or ΔclpX cells the recN be that ΔclpX cells cells and This the that ClpXP degradation of cellular proteins PubMed Scopus Google Scholar, Full Text Full Text PDF PubMed Scopus Google Scholar, 2000; PubMed Scopus Google and therefore the of proteins the of ΔclpX cells to of cytoplasmic RecN aggregates by ClpXP is important for cell viability. of and recN cells. the of wild-type ΔclpX and cells after or of to of and cells after or of to In was as are of at cells are to environmental stimuli such as heat shock or DNA to or after release is for cell by is an of response This study that RecN protein is by the ClpXP protease in a dependent the at the C terminus of RecN. In DNA RecN both and cytoplasmic of the is by DNA and degradation of the ClpXP after DNA damage. In ΔclpX cells, cytoplasmic RecN aggregates after the release from DNA damage and are to cell when to in cells with chronic DNA damage. These data that ClpXP is in after the release from DNA damage by the of DNA RecN protein and is therefore for cell in cells with DNA damage. the that is an of DNA damage that cells to of DNA and repair. of the protein and DNA damage tolerance. DNA double strand breaks (DSBs) 2The abbreviations used are: DSB, DNA double strand break; SMC, structural maintenance of chromosome; GFP, green fluorescent protein; LB, Luria Bertani; DAPI, 4′,6-diamidino-2-phenylindole; MMC, mitomycin C. 2The abbreviations used are: DSB, DNA double strand break; SMC, structural maintenance of chromosome; GFP, green fluorescent protein; LB, Luria Bertani; DAPI, 4′,6-diamidino-2-phenylindole; MMC, mitomycin C. are major threats to the genomic integrity of cells. DSBs can be caused by exogenous and endogenous agents such as ionizing radiation and chemical mutagens and by endogenously produced radicals or as a result of replication arrest and/or collapse of the replication fork (1Cox M.M. Goodman M.F. Kreuzer K.N. Sherratt D.J. Sandler S.J. Marians K.J. Nature. 2000; 404: 37-41Crossref PubMed Scopus (861) Google Scholar, 2Kowalczykowski S.C. Trends Biochem. Sci. 2000; 25: 156-165Abstract Full Text Full Text PDF PubMed Scopus (562) Google DSBs are and result in such as genomic and the of DSBs is for cell and for the integrity of the In Escherichia the of is for the of DNA by its S.C. Trends Biochem. Sci. 2000; 25: 156-165Abstract Full Text Full Text PDF PubMed Scopus (562) Google These DNA DNA a the which is can when the is by coli recN has in the of recN that on recN in response to C. PubMed Scopus Google Scholar, PubMed Google of recN in C. PubMed Scopus Google Scholar, PubMed Scopus Google that RecN is of the of in to the of the in recN to PubMed Scopus Google and is that the RecN protein is required for of DSBs PubMed Scopus Google Scholar, PubMed Google was that RecN is required to breaks at a by PubMed Scopus Google Furthermore, recN are to ionizing radiation and mitomycin C to PubMed Scopus Google RecN of be in the of DNA DSBs by RecN an and and and which are of the maintenance of family Full Text Full Text PDF PubMed Scopus Google Scholar, Sci. PubMed Scopus Google proteins in DNA replication and/or by and in and cells. that proteins in cell and DNA and that the of proteins and are by PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, DNA PubMed Scopus Google Scholar, 25: PubMed Scopus Google In that RecN protein is by the cytoplasmic protease ClpXP in a dependent the signal at the C terminus of RecN. Furthermore, that in ΔclpX cells, DNA RecN formed visible aggregates in the cytoplasm that persisted after release from DNA damage and were to cell These data demonstrate that proteolytic removal of RecN aggregates plays an important role in after release from DNA damage and is therefore for cell and for coli and DNA were as by and for Escherichia coli and and and were used of ionizing radiation was with an of and that used in study were with Sci. 2000; PubMed Scopus Google for and were by PubMed Scopus Google was a from PubMed Scopus Google the and of recN was the PubMed Scopus Google RecN was with an green fluorescent protein at its recN or was by the recN or PubMed Scopus Google recN were by and with the wild-type recN of of was by DNA to C cells were to in Luria at was and were at the was by of the on or were after are the of at and degradation were as PubMed Scopus Google were with for at of was to protein were at the and were rapidly in in at and and by were by as PubMed Scopus Google was with the RecN was were with and with of analysis were by was on a PubMed Scopus Google of or are in the and for coli and DNA were as by and for Escherichia coli and and and were used of ionizing radiation was with an of and that used in study were with Sci. 2000; PubMed Scopus Google for and were by PubMed Scopus Google was a from PubMed Scopus Google the and of recN was the PubMed Scopus Google RecN was with an green fluorescent protein at its recN or was by the recN or PubMed Scopus Google recN were by and with the wild-type recN of of was by DNA to C cells were to in Luria at was and were at the was by of the on or were after are the of at In and degradation were as PubMed Scopus Google were with for at of was to protein were at the and were rapidly in in at and and by were by as PubMed Scopus Google was with the RecN was RecN. were with and with of analysis were by was on a PubMed Scopus Google of or are in the in the and of RecN in and cells. were to to DNA DSBs, and RecN was In wild-type cells to ionizing RecN protein was which is with the that coli recN is a member of the C. PubMed Scopus Google Scholar, PubMed Google RecN rapidly to after to ionizing radiation that RecN is in cells that RecN is or that its is in cells. the of RecN in its in cells to by protein of RecN protein rapidly for in the of at after of RecN was rapidly with a half-life of RecN is and has a short half-life a for ClpXP is by by and PubMed Scopus Google Scholar, PubMed Scopus Google RecN be a for or of RecN was in and is for PubMed Scopus Google the the that the of RecN was in the in that RecN is a for a protein with of or to proteolytic and that are to the in cells Full Text PDF PubMed Google Scholar, Full Text PDF PubMed Google Scholar, PubMed Scopus Google RecN is by or the of RecN was in and that RecN was rapidly in the in the ΔclpX Full Text Full Text PDF PubMed Scopus Google that of after DNA damage revealed the and RecN as of the proteins for ClpXP with of the that RecN is a for ClpXP C of RecN for of RecN was in of RecN of or or of RecN. were with to the of RecN and with and the of RecN was by was rapidly after of In contrast, the or or were These that the of RecN is for degradation of that short degradation the or C terminus of its protein PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, Full Text Full Text PDF PubMed Scopus Google of the degradation is an that is to the C terminus of when PubMed Scopus Google the of RecN is to the degradation that the of RecN be a signal for This was by on in the of RecN. both and in RecN were with RecN and were a and from its in was by and was in the of that the of RecN is for degradation of of RecN in with DNA and of RecN were in an and RecN. was from a its and its was that the of a recN and that the of was with that of RecN from its on the wild-type RecN and to in the of was to for of cells foci This result is with that foci in cells with DNA DSBs PubMed Scopus Google the the and revealed that was localized as a discrete on the at the cell in the cytoplasm These foci are in cells as a In RecN foci were in cells that cytoplasmic foci RecN aggregates. This is by the that coli RecN protein protein aggregates PubMed Scopus Google and protein aggregates are to at cell in the cytoplasm in that the of recN is and in cells with DNA damage. of RecN foci in was in cells from the were to to and to to cells were and/or to that cytoplasmic of foci formed in cells to both cytoplasmic and foci formed in cells to and foci were in were in cells with DNA damage These that DNA DSBs of RecN after from foci were in ΔclpX cells. and cells were with for to MMC, and to for at with for cells were and of cells foci at analysis revealed that is after to in wild-type cells, in ΔclpX cells in wild-type cells, and cytoplasmic foci disappeared rapidly and In contrast, in ΔclpX cells, foci disappeared after to MMC, cytoplasmic foci persisted for and were to cells when cell in and Furthermore, persisted in the of cytoplasmic RecN aggregates after release from DNA damage These data that ClpXP cytoplasmic RecN foci in cells with DNA cytoplasmic RecN aggregates after release from cells were with for MMC, and at the after release from cells were by fluorescent the of cell were by with analysis of after release from and the of cells with RecN foci and cytoplasmic RecN of the cells were of after release from DNA damage. or were as in shows and of cells after removal of of RecN by ClpXP for that to protein aggregates can and Nature. PubMed Scopus Google Scholar, PubMed Scopus Google the role of RecN aggregates in and/or DNA in cells with DNA damage was by cell in and ΔclpX cells with cells were with for cell was in cells in wild-type cells, in ΔclpX and wild-type cells when cells were with for of cell was in ΔclpX cells in wild-type cells of cytoplasmic RecN aggregates are for cell the of cells a that RecN or cell of cells was to the of wild-type RecN after of to was the of wild-type RecN after of to These that cytoplasmic RecN aggregates cell in cells with chronic DNA the of the of RecN for degradation by for is that the RecN aggregates in ΔclpX or cells to of RecN protein and/or DNA proteins and with DNA caused by RecN aggregates is to cells with DNA wild-type and ΔclpX cells in the of DNA damage RecN aggregates were in wild-type or ΔclpX cells the recN be that ΔclpX cells cells and This the that ClpXP degradation of cellular proteins PubMed Scopus Google Scholar, Full Text Full Text PDF PubMed Scopus Google Scholar, 2000; PubMed Scopus Google and therefore the of proteins the of ΔclpX cells to of cytoplasmic RecN aggregates by ClpXP is important for cell viability. of and recN cells. the of wild-type ΔclpX and cells after or of to of and cells after or of to In was as are of at cells are to environmental stimuli such as heat shock or DNA to or after release is for cell by is an of response This study that RecN protein is by the ClpXP protease in a dependent the at the C terminus of RecN. In DNA RecN both and cytoplasmic of the is by DNA and degradation of the ClpXP after DNA damage. In ΔclpX cells, cytoplasmic RecN aggregates after the release from DNA damage and are to cell when to in cells with chronic DNA damage. These data that ClpXP is in after the release from DNA damage by the of DNA RecN protein and is therefore for cell in cells with DNA damage. the that is an of DNA damage that cells to of DNA and repair. of the protein and DNA damage tolerance. RecN in the and of RecN in and cells. were to to DNA DSBs, and RecN was In wild-type cells to ionizing RecN protein was which is with the that coli recN is a member of the C. PubMed Scopus Google Scholar, PubMed Google RecN rapidly to after to ionizing radiation that RecN is in cells that RecN is or that its is in cells. the of RecN in its in cells to by protein of RecN protein rapidly for in the of at after of RecN was rapidly with a half-life of RecN is and has a short half-life RecN a for ClpXP is by by and PubMed Scopus Google Scholar, PubMed Scopus Google RecN be a for or of RecN was in and is for PubMed Scopus Google the the that the of RecN was in the in that RecN is a for a protein with of or to proteolytic and that are to the in cells Full Text PDF PubMed Google Scholar, Full Text PDF PubMed Google Scholar, PubMed Scopus Google RecN is by or the of RecN was in and that RecN was rapidly in the in the ΔclpX Full Text Full Text PDF PubMed Scopus Google that of after DNA damage revealed the and RecN as of the proteins for ClpXP with of the that RecN is a for ClpXP C of RecN for of RecN was in of RecN of or or of RecN. were with to the of RecN and with and the of RecN was by was rapidly after of In contrast, the or or were These that the of RecN is for degradation of RecN. that short degradation the or C terminus of its protein PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, Full Text Full Text PDF PubMed Scopus Google of the degradation is an that is to the C terminus of when PubMed Scopus Google the of RecN is to the degradation that the of RecN be a signal for This was by on in the of RecN. both and in RecN were with RecN and were a and from its in was by and was in the of that the of RecN is for degradation of RecN. of RecN in with DNA and of RecN were in an and RecN. was from a its and its was that the of a recN and that the of was with that of RecN from its on the wild-type RecN and to in the of was to for of cells foci This result is with that foci in cells with DNA DSBs PubMed Scopus Google the the and revealed that was localized as a discrete on the at the cell in the cytoplasm These foci are in cells as a In RecN foci were in cells that cytoplasmic foci RecN aggregates. This is by the that coli RecN protein protein aggregates PubMed Scopus Google and protein aggregates are to at cell in the cytoplasm in that the of recN is and in cells with DNA damage. of RecN foci in was in cells from the were to to and to to cells were and/or to that cytoplasmic of foci formed in cells to both cytoplasmic and foci formed in cells to and foci were in were in cells with DNA damage These that DNA DSBs of RecN after from foci were in ΔclpX cells. and cells were with for to MMC, and to for at with for cells were and of cells foci at analysis revealed that is after to in wild-type cells, in ΔclpX cells in wild-type cells, and cytoplasmic foci disappeared rapidly and In contrast, in ΔclpX cells, foci disappeared after to MMC, cytoplasmic foci persisted for and were to cells when cell in and Furthermore, persisted in the of cytoplasmic RecN aggregates after release from DNA damage These data that ClpXP cytoplasmic RecN foci in cells with DNA damage. of RecN by ClpXP for that to protein aggregates can and Nature. PubMed Scopus Google Scholar, PubMed Scopus Google the role of RecN aggregates in and/or DNA in cells with DNA damage was by cell in and ΔclpX cells with cells were with for cell was in cells in wild-type cells, in ΔclpX and wild-type cells when cells were with for of cell was in ΔclpX cells in wild-type cells of cytoplasmic RecN aggregates are for cell the of cells a that RecN or cell of cells was to the of wild-type RecN after of to was the of wild-type RecN after of to These that cytoplasmic RecN aggregates cell in cells with chronic DNA the of the of RecN for degradation by for is that the RecN aggregates in ΔclpX or cells to of RecN protein and/or DNA proteins and with DNA caused by RecN aggregates is to cells with DNA wild-type and ΔclpX cells in the of DNA damage RecN aggregates were in wild-type or ΔclpX cells the recN be that ΔclpX cells cells and This the that ClpXP degradation of cellular proteins PubMed Scopus Google Scholar, Full Text Full Text PDF PubMed Scopus Google Scholar, 2000; PubMed Scopus Google and therefore the of proteins the of ΔclpX cells to cells are to environmental stimuli such as heat shock or DNA to or after release is for cell by is an of response This study that RecN protein is by the ClpXP protease in a dependent the at the C terminus of RecN. In DNA RecN both and cytoplasmic of the is by DNA and degradation of the ClpXP after DNA damage. In ΔclpX cells, cytoplasmic RecN aggregates after the release from DNA damage and are to cell when to in cells with chronic DNA damage. These data that ClpXP is in after the release from DNA damage by the of DNA RecN protein and is therefore for cell in cells with DNA damage. the that is an of DNA damage that cells to of DNA and repair. of the protein and DNA damage tolerance. for and for