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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.


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The blockage of DrNurA on DrHerA stimulation on DrRecJ nuclease activity and DNA binding ability. (A) DrNurA inhibited DrHerA stimulation on DrRecJ nuclease activity. Hundred nanomolar 5′ overhang DNA (annealed by O2 and O3) was used as substrate and digested by 5 nM DrRecJ. Various concentrations of DrNurA were added in the reaction system (HerA hexamer: NurA dimer = 1:1, 1:4, 1:8, 1:16, 1:32). DrRecJ ssDNA nuclease activity was analyzed in the absence or presence of DrHerA (DrHerA ΔN) in molar ratios (RecJ monomer: HerA hexamer = 1:8). (B) NurA inhibit HerA stimulation on RecJ ssDNA binding activity while DrHerA ΔN do not. Hundred nanomolar 10 nt ssDNA was used as substrate for DrRecJ binding. Fivety nanomolar or 100 nM RecJ was used in the binding assays. Eight hundred nanmolar DrHerA (hexamer) or DrHerA ΔN (hexamer), or 4 μM DrNurA (dimer) was added, if necessary.
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Figure 5: The blockage of DrNurA on DrHerA stimulation on DrRecJ nuclease activity and DNA binding ability. (A) DrNurA inhibited DrHerA stimulation on DrRecJ nuclease activity. Hundred nanomolar 5′ overhang DNA (annealed by O2 and O3) was used as substrate and digested by 5 nM DrRecJ. Various concentrations of DrNurA were added in the reaction system (HerA hexamer: NurA dimer = 1:1, 1:4, 1:8, 1:16, 1:32). DrRecJ ssDNA nuclease activity was analyzed in the absence or presence of DrHerA (DrHerA ΔN) in molar ratios (RecJ monomer: HerA hexamer = 1:8). (B) NurA inhibit HerA stimulation on RecJ ssDNA binding activity while DrHerA ΔN do not. Hundred nanomolar 10 nt ssDNA was used as substrate for DrRecJ binding. Fivety nanomolar or 100 nM RecJ was used in the binding assays. Eight hundred nanmolar DrHerA (hexamer) or DrHerA ΔN (hexamer), or 4 μM DrNurA (dimer) was added, if necessary.

Mentions: DrNurA is another DrHerA interaction partner with nuclease activity, which can be stimulated by DrHerA (Cheng et al., 2015). Given that both DrNurA and DrRecJ possess 5′-3′ ssDNA exonuclease activity and have physical/functional relationships with DrHerA, we were particularly interested in the possible interplay of these three proteins. In order to mimic the 3′ end resection process in vivo, a 5′ overhanging DNA substrate was used. The digestion efficiency of DrRecJ was highly elevated in the presence of DrHerA (Figure 5A, lane 3). However, the addition of catalytic inactive DrNurA mutant (D53A) impaired the stimulation (Figure 5A, lanes 4–8). DrHerAΔN, which still forms a hexametric ATPase but no longer interacts with NurA, could also stimulate the nuclease activity of DrRecJ (Figure 5A, lane 9). The addition of equal amount of DrNurA (D53A) could not inhibit the stimulation (Figure 5A, lanes 10–14). These results suggest DrNurA could competitively bind to DrHerA, thus reducing the stimulation of DrRecJ nuclease activity. However, the DrRecJ and DrNurA appear to interact with DrHerA with different sites because DrHerAΔN could still interact with DrRecJ (Supplemental Figure S5A). Moreover, pull down assays were carried out among DrRecJ, DrHerA, and DrNurA, showing co-binding band of these three proteins (Supplemental Figure S5B, lane 7). Therefore, DrNurA compete with DrRecJ for DrHerA binding is not likely. The addition of DrNurA decreased DrHerA but not DrHerAΔN stimulation of DrRecJ on DNA binding (Figure 5B), indicating that the co-binding of DrNurA on RecJ-HerA reduced the substrate affinity or digestion of RecJ. On the other hand, overwhelming DrNurA strongly inhibits the intrinsic nuclease activity of DrRecJ even if DrHerA or DrHerAΔN were present (Figure 5A, lanes 8 and 14). Reactions without DrHerA were also carried out and showed that high concentrations of DrNurA could inhibit DrRecJ nuclease activity (Supplemental Figure S5C). Moreover, catalytic inactive DrRecJ also inhibits DrHerA stimulation on DrNurA nuclease activity (Supplemental Figure S5D). These results suggest that, in addition to reduce DrHerA stimulation of DrRecJ activity, DrNurA also has weak substrate competition activity with RecJ.


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)

The blockage of DrNurA on DrHerA stimulation on DrRecJ nuclease activity and DNA binding ability. (A) DrNurA inhibited DrHerA stimulation on DrRecJ nuclease activity. Hundred nanomolar 5′ overhang DNA (annealed by O2 and O3) was used as substrate and digested by 5 nM DrRecJ. Various concentrations of DrNurA were added in the reaction system (HerA hexamer: NurA dimer = 1:1, 1:4, 1:8, 1:16, 1:32). DrRecJ ssDNA nuclease activity was analyzed in the absence or presence of DrHerA (DrHerA ΔN) in molar ratios (RecJ monomer: HerA hexamer = 1:8). (B) NurA inhibit HerA stimulation on RecJ ssDNA binding activity while DrHerA ΔN do not. Hundred nanomolar 10 nt ssDNA was used as substrate for DrRecJ binding. Fivety nanomolar or 100 nM RecJ was used in the binding assays. Eight hundred nanmolar DrHerA (hexamer) or DrHerA ΔN (hexamer), or 4 μM DrNurA (dimer) was added, if necessary.
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Figure 5: The blockage of DrNurA on DrHerA stimulation on DrRecJ nuclease activity and DNA binding ability. (A) DrNurA inhibited DrHerA stimulation on DrRecJ nuclease activity. Hundred nanomolar 5′ overhang DNA (annealed by O2 and O3) was used as substrate and digested by 5 nM DrRecJ. Various concentrations of DrNurA were added in the reaction system (HerA hexamer: NurA dimer = 1:1, 1:4, 1:8, 1:16, 1:32). DrRecJ ssDNA nuclease activity was analyzed in the absence or presence of DrHerA (DrHerA ΔN) in molar ratios (RecJ monomer: HerA hexamer = 1:8). (B) NurA inhibit HerA stimulation on RecJ ssDNA binding activity while DrHerA ΔN do not. Hundred nanomolar 10 nt ssDNA was used as substrate for DrRecJ binding. Fivety nanomolar or 100 nM RecJ was used in the binding assays. Eight hundred nanmolar DrHerA (hexamer) or DrHerA ΔN (hexamer), or 4 μM DrNurA (dimer) was added, if necessary.
Mentions: DrNurA is another DrHerA interaction partner with nuclease activity, which can be stimulated by DrHerA (Cheng et al., 2015). Given that both DrNurA and DrRecJ possess 5′-3′ ssDNA exonuclease activity and have physical/functional relationships with DrHerA, we were particularly interested in the possible interplay of these three proteins. In order to mimic the 3′ end resection process in vivo, a 5′ overhanging DNA substrate was used. The digestion efficiency of DrRecJ was highly elevated in the presence of DrHerA (Figure 5A, lane 3). However, the addition of catalytic inactive DrNurA mutant (D53A) impaired the stimulation (Figure 5A, lanes 4–8). DrHerAΔN, which still forms a hexametric ATPase but no longer interacts with NurA, could also stimulate the nuclease activity of DrRecJ (Figure 5A, lane 9). The addition of equal amount of DrNurA (D53A) could not inhibit the stimulation (Figure 5A, lanes 10–14). These results suggest DrNurA could competitively bind to DrHerA, thus reducing the stimulation of DrRecJ nuclease activity. However, the DrRecJ and DrNurA appear to interact with DrHerA with different sites because DrHerAΔN could still interact with DrRecJ (Supplemental Figure S5A). Moreover, pull down assays were carried out among DrRecJ, DrHerA, and DrNurA, showing co-binding band of these three proteins (Supplemental Figure S5B, lane 7). Therefore, DrNurA compete with DrRecJ for DrHerA binding is not likely. The addition of DrNurA decreased DrHerA but not DrHerAΔN stimulation of DrRecJ on DNA binding (Figure 5B), indicating that the co-binding of DrNurA on RecJ-HerA reduced the substrate affinity or digestion of RecJ. On the other hand, overwhelming DrNurA strongly inhibits the intrinsic nuclease activity of DrRecJ even if DrHerA or DrHerAΔN were present (Figure 5A, lanes 8 and 14). Reactions without DrHerA were also carried out and showed that high concentrations of DrNurA could inhibit DrRecJ nuclease activity (Supplemental Figure S5C). Moreover, catalytic inactive DrRecJ also inhibits DrHerA stimulation on DrNurA nuclease activity (Supplemental Figure S5D). These results suggest that, in addition to reduce DrHerA stimulation of DrRecJ activity, DrNurA also has weak substrate competition activity with RecJ.

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