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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

DrHerA enhanced DrRecJ nuclease activity and ssDNA binding ability. (A) DrHerA enhanced DrRecJ nuclease activity. Hundred nanomolar 10 nt ssDNA was used as substrate for RecJ digestion. DrRecJ ssDNA nuclease activity was analyzed in the absence or presence of DrHerA in various molar ratios (RecJ monomer: HerA hexamer = 1:1, 1:4, 1:16). RecJ∗ represented the inactive DrRecJ protein DrRecJ (D158A/H159A/H160A). Five nanomolar DrRecJ was used while 40 nM DrRecJΔC was used in the reaction system. (B) Time course experiments for DrHerA enhancement on DrRecJ nuclease activity. DNA hydrolysis by DrRecJ in the presence or absence of DrHerA was analyzed at different time points (2, 5, 10, 20, 30, and 40 min). (C) Steady-state kinetics analyses of DNA hydrolysis by DrRecJ in the presence or absence of DrHerA. The amount of undegraded substrate remaining for each concentration was quantitated and used to calculate the velocity (v) of the reaction, the reciprocal of which was plotted against the reciprocal of substrate concentration (1/[v] versus 1/[S]). Lineweaver–Burk equation was used for the calculation of kinetic parameters. Error bars indicate standard deviation. (D) DrHerA enhanced DrRecJ ssDNA binding activity. Hundred nanomolar 10 nt ssDNA was used as substrate for RecJ binding. The molar ratio: DNA: DrRecJ = 1:1, 1:2, 1:4; DNA: DrRecJΔC = 1:4, 1:8, 1:16; HerA (hexamer): DrRecJ (or DrRecJΔC) = 8: 1.
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Figure 4: DrHerA enhanced DrRecJ nuclease activity and ssDNA binding ability. (A) DrHerA enhanced DrRecJ nuclease activity. Hundred nanomolar 10 nt ssDNA was used as substrate for RecJ digestion. DrRecJ ssDNA nuclease activity was analyzed in the absence or presence of DrHerA in various molar ratios (RecJ monomer: HerA hexamer = 1:1, 1:4, 1:16). RecJ∗ represented the inactive DrRecJ protein DrRecJ (D158A/H159A/H160A). Five nanomolar DrRecJ was used while 40 nM DrRecJΔC was used in the reaction system. (B) Time course experiments for DrHerA enhancement on DrRecJ nuclease activity. DNA hydrolysis by DrRecJ in the presence or absence of DrHerA was analyzed at different time points (2, 5, 10, 20, 30, and 40 min). (C) Steady-state kinetics analyses of DNA hydrolysis by DrRecJ in the presence or absence of DrHerA. The amount of undegraded substrate remaining for each concentration was quantitated and used to calculate the velocity (v) of the reaction, the reciprocal of which was plotted against the reciprocal of substrate concentration (1/[v] versus 1/[S]). Lineweaver–Burk equation was used for the calculation of kinetic parameters. Error bars indicate standard deviation. (D) DrHerA enhanced DrRecJ ssDNA binding activity. Hundred nanomolar 10 nt ssDNA was used as substrate for RecJ binding. The molar ratio: DNA: DrRecJ = 1:1, 1:2, 1:4; DNA: DrRecJΔC = 1:4, 1:8, 1:16; HerA (hexamer): DrRecJ (or DrRecJΔC) = 8: 1.

Mentions: Since DrHerA could directly interact with DrRecJ, we tested the nuclease activity of DrRecJ in the presence and absence of DrHerA. DrHerA was pre-incubated with DrRecJ to allow the formation of DrRecJ–DrHerA complexes prior to the addition of 10 nt 5′ FAM-labeled ssDNA. The reaction was initiated by the addition of 0.1 mM Mn2+. Along with adding increasing amounts of DrHerA, increasing amounts of processed substrate (1 nt band) was observed, indicating that DrRecJ activity could be stimulated by DrHerA in vitro (Figure 4A). Such stimulations were further confirmed by time course experiments (Figure 4B). No obvious stimulation of DrRecJΔC was observed (Figure 4A), indicating that such stimulation came from direct interactions between DrRecJ-CTD and DrHerA. Stimulations by DrHerA were also observed using other DNA substrates such as longer ssDNA (46 nt) and 5′ overhanging DNA (Supplemental Figure S4A). Furthermore, DrHerA showed no stimulation of the D. radiodurans DrRecJ-like protein (Dr_0826) or EcRecJ activity (Supplemental Figure S4B), implying this stimulation is protein-specific and species-specific.


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)

DrHerA enhanced DrRecJ nuclease activity and ssDNA binding ability. (A) DrHerA enhanced DrRecJ nuclease activity. Hundred nanomolar 10 nt ssDNA was used as substrate for RecJ digestion. DrRecJ ssDNA nuclease activity was analyzed in the absence or presence of DrHerA in various molar ratios (RecJ monomer: HerA hexamer = 1:1, 1:4, 1:16). RecJ∗ represented the inactive DrRecJ protein DrRecJ (D158A/H159A/H160A). Five nanomolar DrRecJ was used while 40 nM DrRecJΔC was used in the reaction system. (B) Time course experiments for DrHerA enhancement on DrRecJ nuclease activity. DNA hydrolysis by DrRecJ in the presence or absence of DrHerA was analyzed at different time points (2, 5, 10, 20, 30, and 40 min). (C) Steady-state kinetics analyses of DNA hydrolysis by DrRecJ in the presence or absence of DrHerA. The amount of undegraded substrate remaining for each concentration was quantitated and used to calculate the velocity (v) of the reaction, the reciprocal of which was plotted against the reciprocal of substrate concentration (1/[v] versus 1/[S]). Lineweaver–Burk equation was used for the calculation of kinetic parameters. Error bars indicate standard deviation. (D) DrHerA enhanced DrRecJ ssDNA binding activity. Hundred nanomolar 10 nt ssDNA was used as substrate for RecJ binding. The molar ratio: DNA: DrRecJ = 1:1, 1:2, 1:4; DNA: DrRecJΔC = 1:4, 1:8, 1:16; HerA (hexamer): DrRecJ (or DrRecJΔC) = 8: 1.
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Figure 4: DrHerA enhanced DrRecJ nuclease activity and ssDNA binding ability. (A) DrHerA enhanced DrRecJ nuclease activity. Hundred nanomolar 10 nt ssDNA was used as substrate for RecJ digestion. DrRecJ ssDNA nuclease activity was analyzed in the absence or presence of DrHerA in various molar ratios (RecJ monomer: HerA hexamer = 1:1, 1:4, 1:16). RecJ∗ represented the inactive DrRecJ protein DrRecJ (D158A/H159A/H160A). Five nanomolar DrRecJ was used while 40 nM DrRecJΔC was used in the reaction system. (B) Time course experiments for DrHerA enhancement on DrRecJ nuclease activity. DNA hydrolysis by DrRecJ in the presence or absence of DrHerA was analyzed at different time points (2, 5, 10, 20, 30, and 40 min). (C) Steady-state kinetics analyses of DNA hydrolysis by DrRecJ in the presence or absence of DrHerA. The amount of undegraded substrate remaining for each concentration was quantitated and used to calculate the velocity (v) of the reaction, the reciprocal of which was plotted against the reciprocal of substrate concentration (1/[v] versus 1/[S]). Lineweaver–Burk equation was used for the calculation of kinetic parameters. Error bars indicate standard deviation. (D) DrHerA enhanced DrRecJ ssDNA binding activity. Hundred nanomolar 10 nt ssDNA was used as substrate for RecJ binding. The molar ratio: DNA: DrRecJ = 1:1, 1:2, 1:4; DNA: DrRecJΔC = 1:4, 1:8, 1:16; HerA (hexamer): DrRecJ (or DrRecJΔC) = 8: 1.
Mentions: Since DrHerA could directly interact with DrRecJ, we tested the nuclease activity of DrRecJ in the presence and absence of DrHerA. DrHerA was pre-incubated with DrRecJ to allow the formation of DrRecJ–DrHerA complexes prior to the addition of 10 nt 5′ FAM-labeled ssDNA. The reaction was initiated by the addition of 0.1 mM Mn2+. Along with adding increasing amounts of DrHerA, increasing amounts of processed substrate (1 nt band) was observed, indicating that DrRecJ activity could be stimulated by DrHerA in vitro (Figure 4A). Such stimulations were further confirmed by time course experiments (Figure 4B). No obvious stimulation of DrRecJΔC was observed (Figure 4A), indicating that such stimulation came from direct interactions between DrRecJ-CTD and DrHerA. Stimulations by DrHerA were also observed using other DNA substrates such as longer ssDNA (46 nt) and 5′ overhanging DNA (Supplemental Figure S4A). Furthermore, DrHerA showed no stimulation of the D. radiodurans DrRecJ-like protein (Dr_0826) or EcRecJ activity (Supplemental Figure S4B), implying this stimulation is protein-specific and species-specific.

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