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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

Interaction assays between DrRecJ and DrHerA. (A) Far western blotting assays. The table above represents the orders of different proteins dotted on the NC membranes. Each membrane was incubated with different proteins and checked by anti-RecJ or anti-HerA antibody. (B) Pull down assays. Four micro liter 0.5 mM RecJ, RecJΔC or RecJ-core (with lysozyme as control) was incubated with His-DrHerA bound Ni-NTA beads and washed until input control be washed off, and analyzed by 12% SDS-PAGE. Lane 1: His-DrHerA; Lane 2: DrRecJ + lysozyme (input control); Lane 3: DrRecJ pulled by His-DrHerA; Lane 4: DrRecJΔC + lysozyme (input control); Lane 5: DrRecJΔC pulled by His-DrHerA; Lane 6: DrRecJ-core + lysozyme (input control); Lane 7: DrRecJ-core pulled by His-DrHerA.
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Figure 3: Interaction assays between DrRecJ and DrHerA. (A) Far western blotting assays. The table above represents the orders of different proteins dotted on the NC membranes. Each membrane was incubated with different proteins and checked by anti-RecJ or anti-HerA antibody. (B) Pull down assays. Four micro liter 0.5 mM RecJ, RecJΔC or RecJ-core (with lysozyme as control) was incubated with His-DrHerA bound Ni-NTA beads and washed until input control be washed off, and analyzed by 12% SDS-PAGE. Lane 1: His-DrHerA; Lane 2: DrRecJ + lysozyme (input control); Lane 3: DrRecJ pulled by His-DrHerA; Lane 4: DrRecJΔC + lysozyme (input control); Lane 5: DrRecJΔC pulled by His-DrHerA; Lane 6: DrRecJ-core + lysozyme (input control); Lane 7: DrRecJ-core pulled by His-DrHerA.

Mentions: That DrRecJΔC could not fully compensate for the mutant defect also promotes us to find out whether the DrRecJ-CTD participates in the interactions with other important proteins in vivo. Total protein extracted from D. radiodurans was incubated with anti-DrRecJ antibody bound protein G beads. The coimmunoprecipitated proteins were concentrated and analyzed by SDS-PAGE (Supplemental Figure S3), followed by identification using mass spectrometry. In addition to DrSSB, DrHerA was also identified as a potential interaction partner of DrRecJ. A direct interaction between DrRecJ and DrHerA was confirmed by far western blotting assays. No interactions between DrHerA-DrRecJΔC, DrHerA-DrRecJ-core, or DrHerA-EcRecJ were observed (Figure 3A), suggesting that the CTD of DrRecJ was the major interaction site. Pull-down assays were also carried out. HerA protein with N-terminal His-tag (His-HerA) was incubated with Ni-NTA beads. The full length DrRecJ rather than truncated DrRecJ (DrRecJΔC or DrRecJ-core) was pulled down by His-HerA (Figure 3B), indicating that DrRecJ and DrHerA interact with each other through the CTD of DrRecJ.


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)

Interaction assays between DrRecJ and DrHerA. (A) Far western blotting assays. The table above represents the orders of different proteins dotted on the NC membranes. Each membrane was incubated with different proteins and checked by anti-RecJ or anti-HerA antibody. (B) Pull down assays. Four micro liter 0.5 mM RecJ, RecJΔC or RecJ-core (with lysozyme as control) was incubated with His-DrHerA bound Ni-NTA beads and washed until input control be washed off, and analyzed by 12% SDS-PAGE. Lane 1: His-DrHerA; Lane 2: DrRecJ + lysozyme (input control); Lane 3: DrRecJ pulled by His-DrHerA; Lane 4: DrRecJΔC + lysozyme (input control); Lane 5: DrRecJΔC pulled by His-DrHerA; Lane 6: DrRecJ-core + lysozyme (input control); Lane 7: DrRecJ-core pulled by His-DrHerA.
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Figure 3: Interaction assays between DrRecJ and DrHerA. (A) Far western blotting assays. The table above represents the orders of different proteins dotted on the NC membranes. Each membrane was incubated with different proteins and checked by anti-RecJ or anti-HerA antibody. (B) Pull down assays. Four micro liter 0.5 mM RecJ, RecJΔC or RecJ-core (with lysozyme as control) was incubated with His-DrHerA bound Ni-NTA beads and washed until input control be washed off, and analyzed by 12% SDS-PAGE. Lane 1: His-DrHerA; Lane 2: DrRecJ + lysozyme (input control); Lane 3: DrRecJ pulled by His-DrHerA; Lane 4: DrRecJΔC + lysozyme (input control); Lane 5: DrRecJΔC pulled by His-DrHerA; Lane 6: DrRecJ-core + lysozyme (input control); Lane 7: DrRecJ-core pulled by His-DrHerA.
Mentions: That DrRecJΔC could not fully compensate for the mutant defect also promotes us to find out whether the DrRecJ-CTD participates in the interactions with other important proteins in vivo. Total protein extracted from D. radiodurans was incubated with anti-DrRecJ antibody bound protein G beads. The coimmunoprecipitated proteins were concentrated and analyzed by SDS-PAGE (Supplemental Figure S3), followed by identification using mass spectrometry. In addition to DrSSB, DrHerA was also identified as a potential interaction partner of DrRecJ. A direct interaction between DrRecJ and DrHerA was confirmed by far western blotting assays. No interactions between DrHerA-DrRecJΔC, DrHerA-DrRecJ-core, or DrHerA-EcRecJ were observed (Figure 3A), suggesting that the CTD of DrRecJ was the major interaction site. Pull-down assays were also carried out. HerA protein with N-terminal His-tag (His-HerA) was incubated with Ni-NTA beads. The full length DrRecJ rather than truncated DrRecJ (DrRecJΔC or DrRecJ-core) was pulled down by His-HerA (Figure 3B), indicating that DrRecJ and DrHerA interact with each other through the CTD of DrRecJ.

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