RNase T is related to dnaQ and family C DNA polymerases

Sir, In our continuing interest in the evolution of DNA polymerase superfamilies, we have conducted homology searches for the editing 3′ to 5′ exonuclease domains of DNA polymerases (Huang, Braithwaite and Ito, 1997, FEBS Lett400: 94–98). Very recently, we learned that Mian has carried out comparative sequence analyses of ribonuclease families (Mian, 1997, Nucleic Acids Res25: 3187–3195). Her study showed that the ribonuclease D family has significant sequence homology with the 3′ to 5′ exonuclease domain of family A DNA polymerases of which E. coli DNA polymerase I is the prototype. Although our results are similar to those of Mian, we found an additional homology between ribonuclease and DNA polymerases that could be significant. As shown in Fig. 1, the RNase T family is closely related to dnaQ proteins from Gram-negative bacteria and DNA polymerase III proteins from Gram-positive bacteria. . Multiple sequence alignment of the RNase T family with dnaQ proteins and family C DNA polymerases. Possible metal-binding ligands are indicated by the arrows and labelled L1-L4 (see text). The 3′-Exo IIIC motif of family C DNA polymerases, HxAxxD, is different from the YxxxD motif common to family A and family B DNA polymerases (Huang et al., 1997, FEBS Lett400: 94–98). To clearly show this difference we have labelled the motif 3′-Exo IIIC for family C and 3′-Exo III for families A and B. Accession numbers: E. coli RNase T (P30014), Vibrio parahaemolyticus RNase T (P46232), Haemophilus influenzae RNase T (P44639), E. coli dna Q (P03007), Salmonella typhimurium dna Q (Q56055/P14566), Haemophilus influenzae dna Q (P43745), Buchnera aphidicola dna Q (U77464), Staphylococcus aureus DNA pol III (D86727/D45368), Bacillus subtilis DNA pol III (P13267), Mycoplasma genitalium DNA pol III (P47277), Mycoplasma pulmonis DNA pol III (P47729). The multiple sequence alignment was performed using the PILEUP program from GCG followed by manual adjustments. RNase T, reported first in 1984 (Deutscher, Marlor and Zaniewski,1984, Proc Natl Acad Sci USA, 81: 4290–4293), is one of seven distinct exoribonucleases that have been identified in eubacteria (Deutscher, 1990, Prog Nucleic Acids Res Mol Biol39: 209–240). All of these exoribonucleases catalyse RNA hydrolysis in the 3′ to 5′ direction. It is known that RNase T is responsible for the removal of the 3′ terminal AMP residue of tRNA during the end-turnover process (Huang and Deutscher, 1992, J Biol Chem267: 25609–25613). The dnaQ gene specifies the ε-subunit of DNA polymerase III holoenzyme that has proofreading 3′ to 5′ exonuclease activity (Scheuermann and Echols, 1984, Proc Natl Acad Sci USA81: 7747–7751). Based on the X-ray crystallographic data and site-directed mutagenesis studies of the Klenow fragment of E. coli DNA polymerase I, the two metal ion catalysis mechanism has been proposed for the 3′ to 5′ exonuclease activity (Joyce and Steitz, 1994, Annu Rev Biochem63: 777–822). These two metal ions are co-ordinated by four carboxylic amino acids (Beese and Steitz, 1991, EMBO J10: 25–33) As shown in Fig. 1, all four amino acid ligands indicated by the arrows (L1–L4) are conserved in these proteins. Although structural data on the dna Q or family C DNA polymerases are currently not available, site-directed mutagenesis studies of the B. subtilis DNA polymerase III indicated that amino acid residues H and D in the 3′-Exo IIIC motif are critical for 3′ to 5′ exonuclease activity (Barnes et al., 1995, Gene, 165: 45–50). The RNase T family resemble closely those of the RNase D family in sizes and catalytic properties, although the primary sequences are not very highly conserved among these family members. If one accepts the view that the ‘RNA World’ preceded the ‘DNA World’, one possible evolutionary scenario would be that these RNase families were diverted before the origin of self-correcting DNA polymerases. Thus, it is possible that these RNase D and T families are the progenitors of the editing 3′ to 5′ exonuclease domains of the DNA polymerases.