3'-5' Exonucleolytic activity of DNA polymerases: structural features that allow kinetic discrimination between ribo- and deoxyribonucleotide residues.
We have determined rates for the excision of nucleotides from the 3' termini of chimeric DNA-RNA oligonucleotides using the Klenow fragment (KF) and two other DNA polymerases, from phages T4 and T7. For these studies, we synthesized DNA-RNA chimeric oligonucleotides with RNA residues in defined positions. When a ribonucleotide residue was placed at the 3' terminus, all three DNA polymerases removed it at the same rate as they did for substrates composed solely of deoxynucleotide residues. There was a decrease in the excision rate, however, when a ribonucleotide residue was located at the second or third position from the 3' terminus. When both the second and third positions were occupied by ribonucleotide residues, the excision rate for the 3' terminal nucleotide was reduced even further and was almost identical to the rate observed when the DNA polymerases encountered single-stranded RNA. The magnitude of the effect of ribonucleotide residues on the excision rate was lower when Mn(2+) replaced Mg(2+) as the essential divalent cation. Two KF mutations, Y423A and N420A, selectively affected the excision rates for the chimeric substrates. Specifically, Y423A totally abolished the rate reduction when there was a single ribonucleotide residue immediately preceding the 3' terminus, whereas N420A diminished, but did not eliminate, the rate reduction relative to that of wild-type KF when the single ribonucleotide residue occupied either the second or third position from the 3' terminus. These results are consistent with the structure of a KF-ss DNA complex from which it can be deduced, by modeling, that a 2' OH group on the second sugar from the 3' terminus would sterically clash with the Tyr 423 side chain, and a 2' OH group on the third sugar would clash with the side chain of Asn 420. The corresponding mutations in T4 DNA polymerase did not affect the rate of hydrolysis of the chimeric oligonucleotides. Thus, there appears to be a major difference in the kinetic behavior of KF and T4 DNA polymerase with respect to the exonuclease reaction. These results are discussed with respect to their possible biological relevance to DNA replication.
Lin
T C
TC
Department of Molecular Biophysics and Biochemistry, Yale University, 333 Cedar Street, New Haven, Connecticut 06520, USA.
Wang
C X
CX
Joyce
C M
CM
Konigsberg
W H
WH
D017122:Bacteriophage T4 / Q000201:enzymology / Q000235:genetics; D017123:Bacteriophage T7 / Q000201:enzymology / Q000235:genetics; D004256:DNA Polymerase I / Q000737:chemistry* / Q000235:genetics; D004277:DNA, Single-Stranded / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D012265:Ribonucleotides / Q000737:chemistry*; D013379:Substrate Specificity / Q000235:genetics; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics
D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D012265:Ribonucleotides; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; C034970:bacteriophage T7 induced DNA polymerase; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V