Phe 771 of Escherichia coli DNA polymerase I (Klenow fragment) is the major site for the interaction with the template overhang and the stabilization of the pre-polymerase ternary complex.
To identify the sites in the Klenow fragment of Escherichia coli DNA polymerase I that interact with the ssDNA overhang of the template strand in the pre-polymerase ternary complex, we carried out UV-mediated photo-cross-linking of the enzyme-DNA-dNTP ternary complex. The template strand contained a nine-nucleotide overhang and was radiolabeled at the 5'-end. Since the enzyme-TP-dNTP ternary complex but not the E-TP binary complex is stable at high ionic strengths, the cross-linking was carried out in the presence of 0.5 M NaCl. The cross-linked E-TP-dNTP complex was purified and subjected to trypsin digestion. The radiolabeled TP cross-linked peptide was further purified by DEAE-Sepharose and C18 column chromatography and subjected to amino acid sequencing. The release of radiolabeled DNA during each sequencing cycle was also monitored. The sequencing results as well as the radioactivity release pattern show that F771, contained in a peptide spanning amino acids 759-775 of pol I, is the unequivocal site of the template cross-linking. A qualitative assessment of the cross-linking efficiency of the template overhang containing a TT sequence at different positions in the ternary complex further suggests that the major cross-linking site within the template overhang is at the second and/or third nucleotide. An examination of the F771A mutant enzyme showed that it was able to form the E-TP binary as well as E-TP-dNTP ternary complex; however, it could not cross-link to the template-primer in the ternary complex. Furthermore, the ternary complex with F771A was qualitatively defective and exhibited some salt sensitivity. These results suggest that F771 participates in the stabilization of the pre-polymerase ternary complex.
Srivastava
Aashish
A
Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey Medical School, Newark, New Jersey 07103, USA.
Singh
Kamalendra
K
Modak
Mukund J
MJ
D001665:Binding Sites / Q000235:genetics; D003432:Cross-Linking Reagents; D004256:DNA Polymerase I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D015224:Dideoxynucleosides / Q000494:pharmacology; D004792:Enzyme Precursors / Q000737:chemistry* / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology*; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D012965:Sodium Chloride; D013379:Substrate Specificity / Q000235:genetics; D013698:Templates, Genetic; D014357:Trypsin / Q000378:metabolism; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D017931:DNA Primers; D015224:Dideoxynucleosides; D004792:Enzyme Precursors; D046911:Macromolecular Substances; D012965:Sodium Chloride; D010649:Phenylalanine; D004256:DNA Polymerase I; D014357:Trypsin