We have identified regions in poly(A) polymerases
that interact with ATP. Conditions were established for
efficient cross-linking of recombinant bovine and yeast
poly(A) polymerases to 8-azido-ATP. Mn2+ strongly
stimulated this reaction due to a 50-fold lower
Ki for 8-azido-ATP in the
presence of Mn2+. Mutations of the highly
conserved Asp residues 113, 115, and 167, critical for
metal binding in the catalytic domain of bovine poly(A)
polymerase, led to a strong reduction of cross-linking
efficiency, and Mn2+ no longer stimulated the
reaction. Sites of 8-azido-ATP cross-linking were mapped
in different poly(A) polymerases by CNBr-cleavage and analysis
of tryptic peptides by mass spectroscopy. The main cross-link
in Schizosaccharomyces pombe poly(A) polymerase
could be assigned to the peptide DLELSDNNLLK (amino acids
167–177). Database searches with sequences surrounding
the cross-link site detected significant homologies to
other nucleotidyltransferase families, suggesting a conservation
of the nucleotide-binding fold among these families of
enzymes. Mutations in the region of the “helical
turn motif” (a domain binding the triphosphate moiety
of the nucleotide) and in the suspected nucleotide-binding
helix of bovine poly(A) polymerase impaired ATP binding
and catalysis. The results indicate that ATP is bound in
part by the helical turn motif and in part by a region
that may be a structural analog to the fingers domain found
in many polymerases.