Archaeal DNA helicase HerA interacts with Mre11 homologue and unwinds blunt-ended double-stranded DNA and recombination intermediates.
HerA is a novel family DNA helicases that exist ubiquitously in thermophilic archaea. The genes are linked to homologues of eukaryotic recombination and repair proteins Mre11 and Rad50 in some of the genomes. However, the relationship between HerA and the related proteins is unclear. In this study, a homologue from the hyperthermophilic archaeon Sulfolobus tokodaii (StoHerA) was characterized and physical and functional interactions between StoHerA and StoMre11 (Mre11 from S. tokodaii) were studied. It was found that StoHerA was able to unwind blunt-ended double-stranded DNA (dsDNA), although with lower efficiency. StoHerA was also able to unwind Holliday junction, splayed-arm DNA, as well as 5'- or 3'-overhang with high efficiency. Pull-down and yeast two-hybrid analyses revealed that StoHerA interacted with StoMre11 physically. The helicase activity of StoHerA was stimulated by StoMre11, indicating a functional role of this interaction. In addition, site-directed mutagenesis of StoHerA was performed to analyze functions of conserved residues of StoHerA. Interestingly, mutation of E355 to alanine in Walker B resulted in not only loss of ATPase and DNA helicase activities, but also dsDNA-binding ability, indicating that this residue is involved in the coupling of ATP hydrolysis, dsDNA-binding, and helicase activities.
Zhang
Songtao
S
State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People's Republic of China.
Wei
Tao
T
Hou
Guihua
G
Zhang
Chao
C
Liang
Pengjuan
P
Ni
Jinfeng
J
Sheng
Duohong
D
Shen
Yulong
Y
D000255:Adenosine Triphosphate / Q000378:metabolism; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D004265:DNA Helicases / Q000378:metabolism*; D019641:DNA, Archaeal / Q000235:genetics*; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011995:Recombination, Genetic; D016944:Sulfolobus / Q000201:enzymology*; D020798:Two-Hybrid System Techniques
D019843:Archaeal Proteins; D019641:DNA, Archaeal; D000255:Adenosine Triphosphate; D004247:DNA; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; C415985:Mre11 protein, archaeal; D004265:DNA Helicases