H3TH domain of bacteriophage T3, T4 RNase H, T5-5' nucleases, and homologs.
H3TH (helix-3-turn-helix) domains of bacteriophage T5-5'nuclease (5'-3' exonuclease or T5FEN), bacteriophage T4 RNase H (T4FEN), bacteriophage T3 (T3 phage exodeoxyribonuclease) and other similar 5' nucleases are included in this family. The T5-5'nuclease is a 5'-3' exodeoxyribonuclease that also exhibits endonucleolytic activity on flap structures (branched duplex DNA containing a free single-stranded 5'end). T4 RNase H, which removes the RNA primers that initiate lagging strand fragments, has 5'- 3' exonuclease activity on DNA/DNA and RNA/DNA duplexes and has endonuclease activity on flap or forked DNA structures. Bacteriophage T3 is believed to function in the removal of DNA-linked RNA primers and is essential for phage DNA replication and also necessary for host DNA degradation and phage genetic recombination. These nucleases are members of the structure-specific, 5' nuclease family that catalyzes hydrolysis of DNA duplex-containing nucleic acid structures during DNA replication, repair, and recombination. They contain a PIN (PilT N terminus) domain with a helical arch/clamp region/I domain (not included here) and inserted within the PIN domain is an atypical helix-hairpin-helix-2 (HhH2)-like region. This atypical HhH2 region, the H3TH domain, has an extended loop with at least three turns between the first two helices, and only three of the four helices appear to be conserved. Both the H3TH domain and the helical arch/clamp region are involved in DNA binding. Studies suggest that a glycine-rich loop in the H3TH domain contacts the phosphate backbone of the template strand in the downstream DNA duplex. The nucleases within this family have a carboxylate rich active site that is involved in binding essential divalent metal ion cofactors required for nuclease activity. The first metal binding site (MBS-1) is composed entirely of Asp/Glu residues from the PIN domain, whereas, the second metal binding site (MBS-2) is composed generally of two Asp residues from the PIN domain and two Asp residues from the H3TH domain. In the T5-5'nuclease, structure-specific endonuclease activity requires binding of a single metal ion in the high-affinity, MBS-1, whereas exonuclease activity requires both, the high-affinity, MBS-1 and the low-affinity, MBS-2 to be occupied by a divalent cofactor. The T5-5'nuclease is reported to be able to bind several metal ions including, Mg2+, Mn2+, Zn2+ and Co2+, as co-factors. Together with the helical arch and network of amino acids interacting with metal binding ions, the H3TH region defines a positively charged active-site DNA-binding groove in structure-specific 5' nucleases.
Feature 1:DNA binding site [nucleic acid binding site]
Evidence:
Structure:2IHN; Bacteriophage T4 RNase H binds a fork DNA substrate, contacts at 5.0 A
Comment:Together with the helical arch and network of amino acids interacting with metal binding ions, the H3TH region defines a positively charged active-site DNA-binding groove in structure-specific 5' nucleases.
Comment:Studies suggest that a glycine-rich loop in the H3TH domain contacts the phosphate backbone of the template strand in the downstream DNA duplex.
Comment:Unique to the T4 RNase H H3TH domain, is a larger (approx. 27 residues) loop present between the first two helices.
Jiyao W et al.(2023). "The conserved domain database in 2023.",