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A Novel C-Terminal Domain of RecJ is Critical for Interaction with HerA in Deinococcus radiodurans.

Cheng K, Zhao Y, Chen X, Li T, Wang L, Xu H, Tian B, Hua Y - Front Microbiol (2015)

Bottom Line: DrRecJΔC displayed reduced DNA nuclease activity and DNA binding ability.Opposing growth and MMC-resistance phenotypes between the recJ and nurA mutants were observed.A novel modulation mechanism among DrRecJ, DrHerA, and DrNurA was also suggested.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Chinese Ministry of Agriculture for Nuclear-Agricultural Sciences, Institute of Nuclear-Agricultural Sciences, Zhejiang University Hangzhou, China.

ABSTRACT
Homologous recombination (HR) generates error-free repair products, which plays an important role in double strand break repair and replication fork rescue processes. DNA end resection, the critical step in HR, is usually performed by a series of nuclease/helicase. RecJ was identified as a 5'-3' exonuclease involved in bacterial DNA end resection. Typical RecJ possesses a conserved DHH domain, a DHHA1 domain, and an oligonucleotide/oligosaccharide-binding (OB) fold. However, RecJs from Deinococcus-Thermus phylum, such as Deinococcus radiodurans RecJ (DrRecJ), possess an extra C-terminal domain (CTD), of which the function has not been characterized. Here, we showed that a CTD-deletion of DrRecJ (DrRecJΔC) could not restore drrecJ mutant growth and mitomycin C (MMC)-sensitive phenotypes, indicating that this domain is essential for DrRecJ in vivo. DrRecJΔC displayed reduced DNA nuclease activity and DNA binding ability. Direct interaction was identified between DrRecJ-CTD and DrHerA, which stimulates DrRecJ nuclease activity by enhancing its DNA binding affinity. Moreover, DrNurA nuclease, another partner of DrHerA, inhibited the stimulation of DrHerA on DrRecJ nuclease activity by interaction with DrHerA. Opposing growth and MMC-resistance phenotypes between the recJ and nurA mutants were observed. A novel modulation mechanism among DrRecJ, DrHerA, and DrNurA was also suggested.

No MeSH data available.


Related in: MedlinePlus

Phenotypes of recJ knockout strain and complemented strains. (A) Schematic representations of RecJ/DHH protein superfamily. Each domain is colored ranging from blue at the N terminus to red at the C terminus based on the structure of ttRecJ (PDB code: 2ZXP). Truncations of DrRecJ were constructed according to the domains of EcRecJ and RecJ-like protein. (B) Growth and MMC resistance features of different strains. The diluted bacteria suspensions (treated with 20 ug ml-1 MMC or not) of wild type strains (R1), drrecJ disruptant (JM), JM with drrecJ complemented (JM-J), JM with drrecJΔC complemented (JM-JΔC), JM with drrecJ-core complemented (JM-Jcore), and JM with ecrecJ complemented (JM-ecJ) were grown to the same OD600, spotted on TGY medium and cultured at 30°C and 37°C. (C) The survival fractions of different strains with different MMC doses treatments. The survival fraction curves were plotted using GraphPad Prism 5 software. The data of R1, JM, JM-J, and JM-JΔC were marked with light red circle, light green square, light blue triangle and light hollow inverted triangle, respectively. Data show mean values from three independent experiments and bars depict the standard deviation (SD). (D) Growth curves of different strains at different temperatures. The OD600 value of each strain was measured at different time points. The growth curves were plotted using GraphPad Prism 5 software. Data shown mean values from three independent experiments and bars depict the standard deviation (SD). Up: assay tested at 30°C; down: assay tested at 37°C.
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Figure 1: Phenotypes of recJ knockout strain and complemented strains. (A) Schematic representations of RecJ/DHH protein superfamily. Each domain is colored ranging from blue at the N terminus to red at the C terminus based on the structure of ttRecJ (PDB code: 2ZXP). Truncations of DrRecJ were constructed according to the domains of EcRecJ and RecJ-like protein. (B) Growth and MMC resistance features of different strains. The diluted bacteria suspensions (treated with 20 ug ml-1 MMC or not) of wild type strains (R1), drrecJ disruptant (JM), JM with drrecJ complemented (JM-J), JM with drrecJΔC complemented (JM-JΔC), JM with drrecJ-core complemented (JM-Jcore), and JM with ecrecJ complemented (JM-ecJ) were grown to the same OD600, spotted on TGY medium and cultured at 30°C and 37°C. (C) The survival fractions of different strains with different MMC doses treatments. The survival fraction curves were plotted using GraphPad Prism 5 software. The data of R1, JM, JM-J, and JM-JΔC were marked with light red circle, light green square, light blue triangle and light hollow inverted triangle, respectively. Data show mean values from three independent experiments and bars depict the standard deviation (SD). (D) Growth curves of different strains at different temperatures. The OD600 value of each strain was measured at different time points. The growth curves were plotted using GraphPad Prism 5 software. Data shown mean values from three independent experiments and bars depict the standard deviation (SD). Up: assay tested at 30°C; down: assay tested at 37°C.

Mentions: RecJ is a DNA exonuclease belonging to the RecJ/DHH superfamily of phosphoesterases. Members of this superfamily usually possess a conserved DHH domain (Domain I, motifs A–D) and a DHHA1 domain (Domain II, motifs E and F) (Supplemental Figure S1A). Besides these two representative domains, typical RecJ proteins always possess a conserved OB fold (domain III). Moreover, RecJs from Deinococcus-Thermus phylum have extra conserved CTDs (domain IV) with more than 20% sequential identities (Figure 1A; Supplemental Figure S1B). However, these CTDs are uncharacterized, which have no defined homolog as analyzed by the HHpred online tool.


A Novel C-Terminal Domain of RecJ is Critical for Interaction with HerA in Deinococcus radiodurans.

Cheng K, Zhao Y, Chen X, Li T, Wang L, Xu H, Tian B, Hua Y - Front Microbiol (2015)

Phenotypes of recJ knockout strain and complemented strains. (A) Schematic representations of RecJ/DHH protein superfamily. Each domain is colored ranging from blue at the N terminus to red at the C terminus based on the structure of ttRecJ (PDB code: 2ZXP). Truncations of DrRecJ were constructed according to the domains of EcRecJ and RecJ-like protein. (B) Growth and MMC resistance features of different strains. The diluted bacteria suspensions (treated with 20 ug ml-1 MMC or not) of wild type strains (R1), drrecJ disruptant (JM), JM with drrecJ complemented (JM-J), JM with drrecJΔC complemented (JM-JΔC), JM with drrecJ-core complemented (JM-Jcore), and JM with ecrecJ complemented (JM-ecJ) were grown to the same OD600, spotted on TGY medium and cultured at 30°C and 37°C. (C) The survival fractions of different strains with different MMC doses treatments. The survival fraction curves were plotted using GraphPad Prism 5 software. The data of R1, JM, JM-J, and JM-JΔC were marked with light red circle, light green square, light blue triangle and light hollow inverted triangle, respectively. Data show mean values from three independent experiments and bars depict the standard deviation (SD). (D) Growth curves of different strains at different temperatures. The OD600 value of each strain was measured at different time points. The growth curves were plotted using GraphPad Prism 5 software. Data shown mean values from three independent experiments and bars depict the standard deviation (SD). Up: assay tested at 30°C; down: assay tested at 37°C.
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Related In: Results  -  Collection

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Figure 1: Phenotypes of recJ knockout strain and complemented strains. (A) Schematic representations of RecJ/DHH protein superfamily. Each domain is colored ranging from blue at the N terminus to red at the C terminus based on the structure of ttRecJ (PDB code: 2ZXP). Truncations of DrRecJ were constructed according to the domains of EcRecJ and RecJ-like protein. (B) Growth and MMC resistance features of different strains. The diluted bacteria suspensions (treated with 20 ug ml-1 MMC or not) of wild type strains (R1), drrecJ disruptant (JM), JM with drrecJ complemented (JM-J), JM with drrecJΔC complemented (JM-JΔC), JM with drrecJ-core complemented (JM-Jcore), and JM with ecrecJ complemented (JM-ecJ) were grown to the same OD600, spotted on TGY medium and cultured at 30°C and 37°C. (C) The survival fractions of different strains with different MMC doses treatments. The survival fraction curves were plotted using GraphPad Prism 5 software. The data of R1, JM, JM-J, and JM-JΔC were marked with light red circle, light green square, light blue triangle and light hollow inverted triangle, respectively. Data show mean values from three independent experiments and bars depict the standard deviation (SD). (D) Growth curves of different strains at different temperatures. The OD600 value of each strain was measured at different time points. The growth curves were plotted using GraphPad Prism 5 software. Data shown mean values from three independent experiments and bars depict the standard deviation (SD). Up: assay tested at 30°C; down: assay tested at 37°C.
Mentions: RecJ is a DNA exonuclease belonging to the RecJ/DHH superfamily of phosphoesterases. Members of this superfamily usually possess a conserved DHH domain (Domain I, motifs A–D) and a DHHA1 domain (Domain II, motifs E and F) (Supplemental Figure S1A). Besides these two representative domains, typical RecJ proteins always possess a conserved OB fold (domain III). Moreover, RecJs from Deinococcus-Thermus phylum have extra conserved CTDs (domain IV) with more than 20% sequential identities (Figure 1A; Supplemental Figure S1B). However, these CTDs are uncharacterized, which have no defined homolog as analyzed by the HHpred online tool.

Bottom Line: DrRecJΔC displayed reduced DNA nuclease activity and DNA binding ability.Opposing growth and MMC-resistance phenotypes between the recJ and nurA mutants were observed.A novel modulation mechanism among DrRecJ, DrHerA, and DrNurA was also suggested.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Chinese Ministry of Agriculture for Nuclear-Agricultural Sciences, Institute of Nuclear-Agricultural Sciences, Zhejiang University Hangzhou, China.

ABSTRACT
Homologous recombination (HR) generates error-free repair products, which plays an important role in double strand break repair and replication fork rescue processes. DNA end resection, the critical step in HR, is usually performed by a series of nuclease/helicase. RecJ was identified as a 5'-3' exonuclease involved in bacterial DNA end resection. Typical RecJ possesses a conserved DHH domain, a DHHA1 domain, and an oligonucleotide/oligosaccharide-binding (OB) fold. However, RecJs from Deinococcus-Thermus phylum, such as Deinococcus radiodurans RecJ (DrRecJ), possess an extra C-terminal domain (CTD), of which the function has not been characterized. Here, we showed that a CTD-deletion of DrRecJ (DrRecJΔC) could not restore drrecJ mutant growth and mitomycin C (MMC)-sensitive phenotypes, indicating that this domain is essential for DrRecJ in vivo. DrRecJΔC displayed reduced DNA nuclease activity and DNA binding ability. Direct interaction was identified between DrRecJ-CTD and DrHerA, which stimulates DrRecJ nuclease activity by enhancing its DNA binding affinity. Moreover, DrNurA nuclease, another partner of DrHerA, inhibited the stimulation of DrHerA on DrRecJ nuclease activity by interaction with DrHerA. Opposing growth and MMC-resistance phenotypes between the recJ and nurA mutants were observed. A novel modulation mechanism among DrRecJ, DrHerA, and DrNurA was also suggested.

No MeSH data available.


Related in: MedlinePlus