A study of the mechanism of T4 DNA polymerase with diastereomeric phosphorothioate analogues of deoxyadenosine triphosphate.

P J Romaniuk,F Eckstein

doi:10.1016/s0021-9258(18)34435-1

Abstract

T4 DNA polymerase copolymerizes the SP isomers of 2'-deoxyadenosine 5'-O-(1-thiotriphosphate) and 5'-O-(2-thiotriphosphate) with dTTP onto a poly(d(A-T) template in the presence of various metal ions. The corresponding RP diastereomers are inactive, independent of the metal ion used. The polymer resulting from the polymerization of the SP diastereomer of 2'-deoxyadenosine 5'-O-(1-thiotriphosphate) and dTTP can be degraded by the 5' leads to 3' exonuclease activity of Escherichia coli DNA polymerase I and alkaline phosphatase (Brody, R. S., and Frey, P. A. (1981) Biochemistry 20, 1245-1252) to d(Tp(S)A). This material has the RP configuration as determined by comparison with the RP and SP diastereomers obtained by chemical synthesis and preparative separation by high performance liquid chromatography. This result indicates inversion of configuration at the alpha-phosphorus in the nucleotidyl transfer reaction and is compatible with the absence of a covalent enzyme intermediate.

Highlights

Retention time"'' Hplc retention times in 7.5% CH:ICNin 0.1 M triethylammonium zenesulfonyl chloride (775mg, 2.56 mmol) was added, and the mixture acetate, pH7.0, flow rate of 2.0 ml/min, on a Shandon ODS-Hypersil '' set aside at room temperature for 1h
T4 DNA polymerase copolymerizes the S, isomers of with T4 DNA polymerase which shows that the structureof
After work-up of this reaction the product was dried with pyridine, reactivated with TPS, raphy to give 40 A?+;[u,nits of isomerically pure d(Tp(S)A) witha and condensed with N'@'-dibenzoyldeoxyadenosine

Summary

Retention time"

'' Hplc retention times in 7.5% CH:ICNin 0.1 M triethylammonium zenesulfonyl chloride (775mg, 2.56 mmol) was added, and the mixture acetate, pH7.0, flow rate of 2.0 ml/min, on a Shandon ODS-Hypersil '' set aside at room temperature for 1h. After a 16-h reaction, tlc on silica gel with CHCI;I:CH.IOH(9:1,V/V). T4DNA polymerase (200 units) was added, and the reaction mixture was incubated at 37 "C. After 20 min, silica gel tlc in CHCI,,:CH.IOH(9:1, v/v) indicated complete conversion of starting material (RF0..51) to product (RF0,.30). The product was eluted with CHCI.I:CH.IOH(97:3,v/v) froma 20-g silica gel column. The reaction mixture was purified by chromatography on DEAE-Sephadex A25 (1.5 X 20 cm) eluted witha linear gradient of 0.25 liters each of 0.02 and 0.2 M triethylammonium bicarbonate.20-1111 fractions were collected. Tchoelumn was eluted isocratically with 7.5%acetonitrile in 0.1 M triethylammonium

RESULTS

Initial rates of polymerization fordATP and phosphorothioate analogues

DISCUSSION