Pre-Steady-State Kinetic Analysis of Sequence-Dependent Nucleotide Excision by the 3'-Exonuclease Activity of Bacteriophage T4 DNA Polymerase

Abstract

The effects of local DNA sequence on the proofreading efficiency of wild-type T4 DNA polymerase were examined by measuring the kinetics of removal of the fluorescent nucleotide analog 2-aminopurine deoxynucleoside monophosphate (dAPMP) from primer/templates of defined sequences. The effects of (1) interactions with the 5'-neighboring bases, (2) base pair stability, and (3) G.C content of the surrounding sequences on the pre-steady-state kinetics of dAPMP excision were measured. Rates of excision dAPMP from a primer 3'-terminus located opposite a template T (AP.T base pair) increased, over a 3-fold range, with the 5'-neighbor to AP in the order C < G < T < A. Rates of removal of dAPMP from AP.X base pairs located in the same surrounding sequence increased as AP.T < AP.A < AP.C < AP.G, which correlates with the decrease in the stabilities of these base pairs predicted by Tm measurements. A key finding was that AP was excised at a slower rate when mispaired opposite C located next to four G.C base pairs than when correctly paired opposite T next to four A.T base pairs, suggesting that exonuclease mismatch removal specificities may be enhanced to a much greater extent by instabilities of local primer termini than by specific recognition of incorrect base pairs. In polymerase-initiated reactions, biphasic reaction kinetics were observed for the excision of AP within most but not all sequence contexts. Rates of the rapid phases (30-40 s-1) were relatively insensitive to sequence context. Rapid-phase rates reflect the rate constants for exonucleolytic excision of dAPMP from melted primer termini for both correct and incorrect base pairs and were roughly comparable to rates of removal of dAPMP from single-stranded DNA (65-80 s-1). Rates of the slow phases (3-13 s-1) were dependent on sequence context; the slow phase may reflect the rate of switching from the polymerase to the exonuclease active site, or perhaps the conversion of a primer/template terminus from an annealed to a melted state in the exonuclease active site. These data, using wild-type T4 DNA polymerase and two exonuclease-deficient T4 polymerases, support a model in which exonuclease excision occurs on melted primer 3'-termini for both mismatched and correctly matched primer termini, and where specificity favoring removal of terminally mismatched base pairs is determined by the much larger fraction of melted-out primer 3'-termini for mispairs compared to that for correct pairs.

Catalytic Mechanism of Bacteriophage T4 Rad50 ATP Hydrolysis

“…2-AP has little fluorescence when it is involved with a base-stacked partner; this fluorescence is greatly enhanced when the base stacking is removed. 23 The nuclease rate was estimated for the MR complex with the 2-AP at position 1 in the presence and absence of ATP (Table 5). In the absence of ATP, the WT complex excised the 2-AP at a rate of 6.8 ± 1.6 min −1 .…”

2-Aminopurine fluorescence quenching and lifetimes: Role of base stacking

“…T he nucleotide 2-aminopurine (2AP) has been used as a site-specific probe of nucleic acid structure and dynamics (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11) because it base pairs with cytosine in a wobble configuration (4,5,12) or with thymine in a Watson-Crick geometry (7,11). Thermodynamic measurements of DNAs containing 2AP:C or 2AP:T show that the former pairing is more destabilizing (11,12).…”

“…The distribution of stacked states can be altered by temperature, solvent, flanking bases, and bound protein, and so all of these conditions can be probed by using 2AP fluorescence. Dynamics of stacked bases adjacent to a mutagenic mismatch in DNA (such as 2AP:C and 2AP:T) may be part of the recognition mechanism by replication͞repair enzymes (3,13), so interpretation of 2AP fluorescence data is critical for describing these environments.…”

Synthesis and fluorescence study of 7-azaindole in DNA oligonucleotides replacing a purine base

“…There are mainly two groups of 'bases' synthesized and incorporated into DNA oligomers: those made by minor modification of the natural bases or the derivatives of cyclic aromatic compounds. While the former includes 2-aminopurine [1][2][3][4][5][6][7][8], inosine [9] and isoinosine [9][10][11], 3-and 7-deazaadenine [12][13][14][15], and 3-and 7-deazaguanine [14,16], the latter includes analogs of pteridine [17][18][19] and indole [20][21][22][23][24], benzene [23][24][25][26], naphthalene and pyrene derivatives [24], The first group of compounds, when replacing a natural base in duplex DNA, can still form hydrogen bonds with the base in the opposite strand, while the second group of compounds cannot form any hydrogen bonds.…”

“…The fluorescence properties of the above base analogs have been utilized for studying DNA structure and duplex stability [1,2,10,14,15,22,26], protein-DNA interactions [4][5][6][7][8]13,18,25], or simply as universal base analogs [20,27]. Some of the unusual 'bases' are used as anti-HIV or anti-cancer drugs [28][29][30].…”