The trimeric Hef-associated nuclease HAN is a 3′→5′ exonuclease and is probably involved in DNA repair

Abstract

Nucleases play important roles in nucleic acid metabolism. Some archaea encode a conserved protein known as Hef-associated nuclease (HAN). In addition to its C-terminal DHH nuclease domain, HAN also has three N-terminal domains, including a DnaJ-Zinc-finger, ribosomal protein S1-like, and oligonucleotide/oligosaccharide-binding fold. To further understand HAN’s function, we biochemically characterized the enzymatic properties of HAN from Pyrococcus furiosus (PfuHAN), solved the crystal structure of its DHH nuclease domain, and examined its role in DNA repair. Our results show that PfuHAN is a Mn2+-dependent 3′-exonuclease specific to ssDNA and ssRNA with no activity on blunt and 3′-recessive double-stranded DNA. Domain truncation confirmed that the intrinsic nuclease activity is dependent on the C-terminal DHH nuclease domain. The crystal structure of the DHH nuclease domain adopts a trimeric topology, with each subunit adopting a classical DHH phosphoesterase fold. Yeast two hybrid assay confirmed that the DHH domain interacts with the IDR peptide of Hef nuclease. Knockout of the han gene or its C-terminal DHH nuclease domain in Haloferax volcanii resulted in increased sensitivity to the DNA damage reagent MMS. Our results imply that HAN nuclease might be involved in repairing stalled replication forks in archaea.

Structure and Function of Piezophilic Hyperthermophilic Pyrococcus yayanosii pApase

“…In several DHH superfamily members, such as bacterial NrnA and RecJ, motifs I-IV are involved in coordinating a divalent metal cofactor that is necessary for hydrolyzing the phosphodiester bond [20][21][22]. In PyapApase, residues D10 and D12 in motif I, D59 in motif II, H78 in motif III, and D126 in motif IV (Figures 1C and 4A and Figure S5) are predicted to be mainly responsible for binding Mn 2+ based on the superimposition of the structures of PyapApase and BsNrnA.…”

“…The DHHA1 family possesses a typical conserved motif and contains many members, including the nucleases RecJ [20], NrnA [16], and Hef-associated nuclease (HAN) [21]. In bacteria, the NrnA subfamily shows strong phosphoesterase activity on the 3 phosphoester bond of pAp.…”

Haloferax volcanii —a model archaeon for studying DNA replication and repair

“…Hef and HAN interact with PCNA in T. kodakarensis , although Hef cannot bind both PCNA and HAN simultaneously [ 443 ]. Deletion of han in H. volcanii (HVO_1018; also called recJ3 ) results in increased sensitivity to DNA damage agent MMS, but not to other genotoxic agents such as H 2 O 2 , 4NQO or MMC [ 446 ]. It has been proposed that HAN acts to coordinate the helicase and nuclease activities of Hef during the processing of stalled replication forks [ 110 , 442 ].…”

Archaeal DNA Repair Mechanisms

“…XPB helicase is sometimes found encoded in an operon with a nuclease named Bax1 and these enzyme act in concert to open a DNA bubble and make cuts [125]. In many XPF-encoding species, the 3 -5 exonuclease HAN is often encoded, potentially recapitulating in vitro experiments where XPF and HAN form a functional nuclease complex [126]. Recent biochemical examinations of archaeal XPF, or Hef, have investigated the enzyme in the context of replication restart and Holliday junction formation [120,127,128], but it is possible that XPF performs multiple functions within the cell-including one in an NER pathway.…”