The RecJ2 protein in the thermophilic archaeon Thermoplasma acidophilum is a 3′-5′ exonuclease that associates with a DNA replication complex

Abstract

RecJ/cell division cycle 45 (Cdc45) proteins are widely conserved in the three domains of life, in bacteria, Eukarya, and Archaea. Bacterial RecJ is a 5'-3' exonuclease and functions in DNA repair pathways by using its 5'-3' exonuclease activity. Eukaryotic Cdc45 has no identified enzymatic activity but participates in the CMG complex, so named because it is composed of Cdc45, minichromosome maintenance protein complex (MCM) proteins 2-7, and GINS complex proteins (Sld5, Psf11-3). Eukaryotic Cdc45 and bacterial/archaeal RecJ share similar amino acid sequences and are considered functional counterparts. In Archaea, a RecJ homolog in was shown to associate with GINS and accelerate its nuclease activity and was, therefore, designated GAN (INS-ssociated uclease); however, to date, no archaeal RecJ·MCM·GINS complex has been isolated. The thermophilic archaeon has two RecJ-like proteins, designated TaRecJ1 and TaRecJ2. TaRecJ1 exhibited DNA-specific 5'-3' exonuclease activity, whereas TaRecJ2 had 3'-5' exonuclease activity and preferred RNA over DNA. TaRecJ2, but not TaRecJ1, formed a stable complex with TaGINS in a 2:1 molar ratio. Furthermore, the TaRecJ2·TaGINS complex stimulated activity of TaMCM ( MCM) helicase , and the TaRecJ2·TaMCM·TaGINS complex was also observed However, TaRecJ2 did not interact with TaMCM directly and was not required for the helicase activation These findings suggest that the function of archaeal RecJ in DNA replication evolved divergently from Cdc45 despite conservation of the CMG-like complex formation between Archaea and Eukarya.

Two Archaeal RecJ Nucleases from Methanocaldococcus jannaschii Show Reverse Hydrolysis Polarity: Implication to Their Unique Function in Archaea

“…We selected three other species, Thermoplasma acidophilum , Archeoglobus fulgidus , and Methanococcus voltae , to characterize the enzymatic activities of their RecJ homologs. For T. acidophilum we confirmed the 5′ exonuclease of RecJ1 and the 3′ exonuclease of RecJ2 [ 31 ]. The two RecJ1s from A. fulgidus and M. voltae were also 5′ exonuclease specific on ssDNA, while we could not identify any nuclease activity of their RecJ2s.…”

“…Surprisingly, both MjaRecJs did not form a complex with MjaGINS. However, the work on the RecJs from Thermoplasma acidophilum showed that TacRecJ2 forms a complex with GINS, but TacRecJ1 not [ 31 ]. Our result on MjaRecJ1 is consistent to that of TacRecJ1, but MjaRecJ2 showed the result contrary to TacRecJ2.…”

“…BLAST with the Pyrococcus furiosus RecJ as a query sequence, it identified more than one RecJ gene in some archaea genomes, especially the methane-producing species. Previous works also found out the diversity of archaeal RecJ [ 16 , 31 ]. During the preparation of our manuscript, Ishino and coworker reported the biochemical characterization of two RecJs from Thermoplasma acidophilum [ 31 ].…”

“…Previous works also found out the diversity of archaeal RecJ [ 16 , 31 ]. During the preparation of our manuscript, Ishino and coworker reported the biochemical characterization of two RecJs from Thermoplasma acidophilum [ 31 ]. TacRecJ1 is a ssDNA specific 5′exonuclease, and TacRecJ2 is a 3′ exonuclease on both ssDNA and ssRNA.…”

The archaeal RecJ-like proteins: nucleases and ex-nucleases with diverse roles in replication and repair

“…T. acidophylum, a thermoacidophilic eukaryarcheon of the order Thermoplasmatales, encodes a pair of RecJ-like proteins, designated TaRecJ1 and TaRecJ2 (see Table 1), both of which are members of the RecJ clade that includes TkoGAN (23) E. coli was able to partially rescue the UV sensitivity of a recJ mutant strain, implying at least partial conservation of function across evolution (24).…”