H3TH domains of structure-specific 5' nucleases (or flap endonuclease-1-like) involved in DNA replication, repair, and recombination.
The 5' nucleases of this superfamily are capable of both 5'-3' exonucleolytic activity and cleaving bifurcated or branched DNA, in an endonucleolytic, structure-specific manner, and are involved in DNA replication, repair, and recombination. The superfamily includes the H3TH (helix-3-turn-helix) domains of Flap Endonuclease-1 (FEN1), Exonuclease-1 (EXO1), Mkt1, Gap Endonuclease 1 (GEN1) and Xeroderma pigmentosum complementation group G (XPG) nuclease. Also included are the H3TH domains of the 5'-3' exonucleases of DNA polymerase I and single domain protein homologs, as well as, the bacteriophage T4 RNase H, T5-5'nuclease, and other homologs. These nucleases contain a PIN (PilT N terminus) domain with a helical arch/clamp region/I domain (not included here) and inserted within the C-terminal region of 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. Typically, the nucleases within this superfamily have a carboxylate rich active site that is involved in binding essential divalent metal ion cofactors (i. e., Mg2+, Mn2+, Zn2+, or Co2+) required for nuclease activity. The first metal binding site is composed entirely of Asp/Glu residues from the PIN domain, whereas, the second metal binding site is composed generally of two Asp residues from the PIN domain and one or two Asp residues from the H3TH domain. 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.
Comment:Mkt1 lacks the glycine-rich loop in the H3TH domain which is proposed to facilitate duplex DNA binding.
Jiyao W et al.(2023). "The conserved domain database in 2023.",