Photoaffinity labeling of DNA template-primer binding site in Escherichia coli DNA polymerase I. Identification of involved amino acids.

Abstract:

We have used two self-annealing template-primers (TPs) to covalently cross-link the Klenow fragment of Escherichia coli DNA polymerase I in its polymerase mode. The specificity of cross-linking is demonstrated by the observation that other template-primers, but not the template or primer alone, readily compete with self-annealing TPs. The enzyme-TP covalent complex is catalytically active and can incorporate one nucleotide on the primer terminus of the immobilized template-primer. Using a peptide mapping approach, we have identified a 17-amino acid tryptic peptide spanning residues 759-775 as a major constituent of the TP binding domain. Amino acid sequence analysis further revealed that Ile-765, Tyr-766 in the O-helix and Ser-769, Phe-771 in the O1-helix of the three-dimensional crystal structure of the Klenow fragment constitute the attachment site for TP.

Polymerases:

Topics:

Status:

new topics/pols set partial results complete validated

Results:

No results available for this paper.

Entry validated by:

Log in to edit reference All References

Using Polbase tables:

Sorting:

Tables may be sorted by clicking on any of the column titles. A second click reverses the sort order. <Ctrl> + click on the column titles to sort by more than one column (e.g. family then name).

Filtering:

It is also possible to filter the table by typing into the search box above the table. This will instantly hide lines from the table that do not contain your search text.