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Solution conformations of primer DNA shuttling between the polymerase and 3′‐5′ exonuclease sites of DNA polymerase I
Author(s) -
Datta Kausiki,
Johnson Neil P.,
LiCata Vince J.,
Hippel Peter H.
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.481.7
Subject(s) - primer (cosmetics) , dna polymerase , exonuclease , dna , polymerase , dna clamp , chemistry , biophysics , dna polymerase ii , microbiology and biotechnology , stereochemistry , biochemistry , biology , polymerase chain reaction , gene , reverse transcriptase , organic chemistry
E. coli DNA polymerase I serves as an excellent model for understanding structure‐function relationships in DNAP. To better understand the role of the intrinsic 3'→5' exonuclease (exo) activity of DNAP in controlling the fidelity of DNA synthesis we have examined the equilibrium distribution of the primer terminus between the exo and polymerase (pol) active sites during both template‐directed synthesis and misincorporation processes. By monitoring the low energy (>300 nm) circular dichroism and fluorescence of 2‐aminopurine dimer probes placed at various positions in P rimer‐ T emplate (P‐T) DNA constructs we have been able to confirm structural studies which showed that when the 3′‐end of the primer is bound at the exo site the three residues at primer terminus are significantly 'frayed' beyond the level characteristic of the ss‐DNA conformation. We have also shown that up to four bps of the ds‐DNA at the P‐T junction adopt an A‐form conformation when the primer end occupies the pol site, but not when it is bound at the exo site. This shows that the duplex DNA beyond the P‐T junction plays an important mechanistic role in guiding the primer end to the appropriate active site. In addition we show that even for matched P‐T DNA the primer terminus partitions between the two active sites, and that the fractional occupancy of the two sites depends on the stability of the bps at the P‐T junction and on the presence of divalent metal ions.