Limits...
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

Comparisons of nuclease activity and ssDNA binding ability among DrRecJ, DrRecJΔC and DrRecJ-core. (A) Nuclease activities of DrRecJ, DrRecJΔC and DrRecJ-core. Hundred nanomolar 10 nt ssDNA was used as substrate for RecJ digestion. The molar ratio of ssDNA:DrRecJ (or DrRecJΔC, DrRecJ) = 5:1, 5:4, 5:16, 5:64. (B) ssDNA binding ability of DrRecJ, DrRecJΔC and DrRecJ-core. Hundred nanomolar 10 nt ssDNA was used as substrate for RecJ binding. The molar ratio of ssDNA:RecJ (or DrRecJΔC, DrRecJ) = 1:2, 1:4, 1:8, 1:16.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4663267&req=5

Figure 2: Comparisons of nuclease activity and ssDNA binding ability among DrRecJ, DrRecJΔC and DrRecJ-core. (A) Nuclease activities of DrRecJ, DrRecJΔC and DrRecJ-core. Hundred nanomolar 10 nt ssDNA was used as substrate for RecJ digestion. The molar ratio of ssDNA:DrRecJ (or DrRecJΔC, DrRecJ) = 5:1, 5:4, 5:16, 5:64. (B) ssDNA binding ability of DrRecJ, DrRecJΔC and DrRecJ-core. Hundred nanomolar 10 nt ssDNA was used as substrate for RecJ binding. The molar ratio of ssDNA:RecJ (or DrRecJΔC, DrRecJ) = 1:2, 1:4, 1:8, 1:16.

Mentions: The OB fold of RecJ was confirmed to assist DNA binding in T. thermophilus (Wakamatsu et al., 2010). However, the function of CTD has not been characterized. We purified DrRecJ, DrRecJΔC, and DrRecJ-core (Supplemental Figure S2) and tested their nuclease activities and DNA binding activities respectively. Compared with DrRecJ, both DrRecJ-core and DrRecJΔC showed reduced nuclease activity (Figure 2A). Similarly, both DrRecJ-core and DrRecJΔC showed much weaker ssDNA affinity than full length DrRecJ, which was in agreement with those of the nuclease activity (Figure 2B). These results indicate that the CTD of DrRecJ also contributes to RecJ DNA binding capability.


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)

Comparisons of nuclease activity and ssDNA binding ability among DrRecJ, DrRecJΔC and DrRecJ-core. (A) Nuclease activities of DrRecJ, DrRecJΔC and DrRecJ-core. Hundred nanomolar 10 nt ssDNA was used as substrate for RecJ digestion. The molar ratio of ssDNA:DrRecJ (or DrRecJΔC, DrRecJ) = 5:1, 5:4, 5:16, 5:64. (B) ssDNA binding ability of DrRecJ, DrRecJΔC and DrRecJ-core. Hundred nanomolar 10 nt ssDNA was used as substrate for RecJ binding. The molar ratio of ssDNA:RecJ (or DrRecJΔC, DrRecJ) = 1:2, 1:4, 1:8, 1:16.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4663267&req=5

Figure 2: Comparisons of nuclease activity and ssDNA binding ability among DrRecJ, DrRecJΔC and DrRecJ-core. (A) Nuclease activities of DrRecJ, DrRecJΔC and DrRecJ-core. Hundred nanomolar 10 nt ssDNA was used as substrate for RecJ digestion. The molar ratio of ssDNA:DrRecJ (or DrRecJΔC, DrRecJ) = 5:1, 5:4, 5:16, 5:64. (B) ssDNA binding ability of DrRecJ, DrRecJΔC and DrRecJ-core. Hundred nanomolar 10 nt ssDNA was used as substrate for RecJ binding. The molar ratio of ssDNA:RecJ (or DrRecJΔC, DrRecJ) = 1:2, 1:4, 1:8, 1:16.
Mentions: The OB fold of RecJ was confirmed to assist DNA binding in T. thermophilus (Wakamatsu et al., 2010). However, the function of CTD has not been characterized. We purified DrRecJ, DrRecJΔC, and DrRecJ-core (Supplemental Figure S2) and tested their nuclease activities and DNA binding activities respectively. Compared with DrRecJ, both DrRecJ-core and DrRecJΔC showed reduced nuclease activity (Figure 2A). Similarly, both DrRecJ-core and DrRecJΔC showed much weaker ssDNA affinity than full length DrRecJ, which was in agreement with those of the nuclease activity (Figure 2B). These results indicate that the CTD of DrRecJ also contributes to RecJ DNA binding capability.

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