DNA Polymerase β Ribonucleotide Discrimination: INSERTION, MISINSERTION, EXTENSION, AND CODING

Nisha A Cavanaugh,William A Beard,Samuel H Wilson

doi:10.1074/jbc.m110.132407

Abstract

DNA polymerases must select nucleotides that preserve Watson-Crick base pairing rules and choose substrates with the correct (deoxyribose) sugar. Sugar discrimination represents a great challenge because ribonucleotide triphosphates are present at much higher cellular concentrations than their deoxy-counterparts. Although DNA polymerases discriminate against ribonucleotides, many therapeutic nucleotide analogs that target polymerases have sugar modifications, and their efficacy depends on their ability to be incorporated into DNA. Here, we investigate the ability of DNA polymerase beta to utilize nucleotides with modified sugars. DNA polymerase beta readily inserts dideoxynucleoside triphosphates but inserts ribonucleotides nearly 4 orders of magnitude less efficiently than natural deoxynucleotides. The efficiency of ribonucleotide insertion is similar to that reported for other DNA polymerases. The poor polymerase-dependent insertion represents a key step in discriminating against ribonucleotides because, once inserted, a ribonucleotide is easily extended. Likewise, a templating ribonucleotide has little effect on insertion efficiency or fidelity. In contrast to insertion and extension of a ribonucleotide, the chemotherapeutic drug arabinofuranosylcytosine triphosphate is efficiently inserted but poorly extended. These results suggest that the sugar pucker at the primer terminus plays a crucial role in DNA synthesis; a 3'-endo sugar pucker facilitates nucleotide insertion, whereas a 2'-endo conformation inhibits insertion.

Highlights

DNA polymerases must select nucleotides that preserve Watson-Crick base pairing rules and choose substrates with the correct sugar
In contrast to insertion and extension of a ribonucleotide, the chemotherapeutic drug arabinofuranosylcytosine triphosphate is efficiently inserted but poorly extended. These results suggest that the sugar pucker at the primer terminus plays a crucial role in DNA synthesis; a 3؅-endo sugar pucker facilitates nucleotide insertion, whereas a 2؅-endo conformation inhibits insertion
Insertion and Misinsertion of Dideoxynucleotides—To determine how the structure of the deoxyribose sugar affects nucleotide incorporation by pol ␤, we examined various nucleotide sugars modified at C2Ј and C3Ј of the furanose ring and measured how efficiently pol ␤ inserted them in a single-nucleotide gapped DNA substrate (Fig. 2)

Summary

EXPERIMENTAL PROCEDURES

[␥-32P]ATP was obtained from PerkinElmer Life Sciences. The ddNTPs were purchased from Amersham Biosciences, and araCTP was purchased from Jena Biosciences (Germany). Arg-183 (R183) and O3Ј of the incoming nucleotide hydrogen bond to a nonbridging oxygen on the ␤-phosphate (dashed green lines). The side chain of Tyr-271 hydrogen bonds with the minor groove edge of the primer terminal base (dashed green line). Arg-283 (R283) of ␣-helix N hydrogen bonds with the nucleotide opposite the primer terminus, (n Ϫ 1)t, at O4Ј of the sugar ring. The DNA substrates were has been successfully applied to examine incoming nucleotide annealed (1:1.2:1.2, primer/template/downstream oligonucleobase attributes (size, volume, hydrogen bonding capacity, and tide) as described previously (see Fig. 2A) [15]. This approach has been used to nucleotide gap-filling reactions were determined as described probe triphosphate attributes that influence binding with pol ␤ previously [15]. The results represent the mean Ϯ S.E. of at least two independent determinations

Sugar discriminationa

RESULTS

DISCUSSION

Catalytic efficiencya

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