title: Biochemical and Functional Characterization of the NurA-HerA Complex from Deinococcus radiodurans.
abstract: In archaea, the NurA nuclease and HerA ATPase/helicase, together with the Mre11-Rad50 complex, function in 3' single-stranded DNA (ssDNA) end processing during homologous recombination (HR). However, bacterial homologs of NurA and HerA have not been characterized. From Deinococcus radiodurans, we identified the manganese-dependent 5'-to-3' ssDNA/double-stranded DNA (dsDNA) exonuclease/endonuclease NurA (DrNurA) and the ATPase HerA (DrHerA). These two proteins stimulated each other's activity through direct protein-protein interactions. The N-terminal HAS domain of DrHerA was the key domain for this interaction. Several critical residues of DrNurA and DrHerA were verified by site-directed mutational analysis. Temperature-dependent activity assays confirmed that the two proteins had mesophilic features, with optimum activity temperatures 10 °C to 15 °C higher than their optimum growth temperatures. Knocking out either nurA or herA affected cell proliferation by shortening the growth phase, especially for growth at a high temperature (37 °C). In addition, both mutant strains displayed almost 10-fold-reduced intermolecular recombination efficiency, indicating that DrNurA and DrHerA might be involved in homologous recombination in vivo. However, single- and double-gene deletions did not show significantly decreased radioresistance. Our results confirmed that the biochemical activities of bacterial NurA and HerA proteins were conserved with archaea. Our phenotypical results suggested that these proteins might have different functions in bacteria.
  
  
  OBJECTIVE
  Deinococcus radiodurans NurA (DrNurA) was identified as a manganese-dependent 5'-to-3' ssDNA/dsDNA exonuclease/endonuclease, and Deinococcus radiodurans HerA (DrHerA) was identified as an ATPase. Physical interactions between DrNurA and DrHerA explained mutual stimulation of their activities. The N-terminal HAS domain on DrHerA was identified as the interaction domain. Several essential functional sites on DrNurA and DrHerA were characterized. Both DrHerA and DrNurA showed mesophilic biochemical features, with their optimum activity temperatures 10 °C to 15 °C higher than their optimum growth temperatures in vitro. Knockout of nurA or herA led to abnormal cell proliferation and reduced intermolecular recombination efficiency but no obvious effect on radioresistence.
authors: Key Laboratory of Chinese Ministry of Agriculture for Nuclear-Agricultural Sciences, Institute of Nuclear-Agricultural Sciences, Zhejiang University, Hangzhou, China.
authors: Kaiying
authors: K
authors: Cheng
authors: Key Laboratory of Chinese Ministry of Agriculture for Nuclear-Agricultural Sciences, Institute of Nuclear-Agricultural Sciences, Zhejiang University, Hangzhou, China.
authors: Xuanyi
authors: X
authors: Chen
authors: Agriculture and Food Science School, Zhejiang Agriculture and Forestry University, Zhejiang, Lin'an, China.
authors: Guangzhi
authors: G
authors: Xu
authors: Key Laboratory of Chinese Ministry of Agriculture for Nuclear-Agricultural Sciences, Institute of Nuclear-Agricultural Sciences, Zhejiang University, Hangzhou, China.
authors: Liangyan
authors: L
authors: Wang
authors: Key Laboratory of Chinese Ministry of Agriculture for Nuclear-Agricultural Sciences, Institute of Nuclear-Agricultural Sciences, Zhejiang University, Hangzhou, China.
authors: Hong
authors: H
authors: Xu
authors: Key Laboratory of Chinese Ministry of Agriculture for Nuclear-Agricultural Sciences, Institute of Nuclear-Agricultural Sciences, Zhejiang University, Hangzhou, China.
authors: Su
authors: S
authors: Yang
authors: Key Laboratory of Chinese Ministry of Agriculture for Nuclear-Agricultural Sciences, Institute of Nuclear-Agricultural Sciences, Zhejiang University, Hangzhou, China yezhao@zju.edu.cn yjhua@zju.edu.cn.
authors: Ye
authors: Y
authors: Zhao
authors: Key Laboratory of Chinese Ministry of Agriculture for Nuclear-Agricultural Sciences, Institute of Nuclear-Agricultural Sciences, Zhejiang University, Hangzhou, China yezhao@zju.edu.cn yjhua@zju.edu.cn.
authors: Yuejin
authors: Y
authors: Hua
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D049109:Cell Proliferation; D019295:Computational Biology; D004260:DNA Repair / Q000502:physiology; D004269:DNA, Bacterial / Q000235:genetics; D034301:Deinococcus / Q000378:metabolism*; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic; D006358:Hot Temperature; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009876:Operon; D011487:Protein Conformation
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D000251:Adenosine Triphosphatases