DNA polymerases can misinsert ribonucleotides that lead to genomic instability. DNA polymerase ␤ discourages ribonucleotide insertion with the backbone carbonyl of Tyr-271; alanine substitution of Tyr-271, but not Phe-272, resulted in a >10-fold loss in discrimination. The Y271A mutant also inserted ribonucleotides more efficiently than wild type on a variety of ribonucleoside (rNMP)-containing DNA substrates. Substituting Mn 2؉ for Mg 2؉ decreased sugar discrimination for both wildtype and mutant enzymes primarily by increasing the affinity for rCTP. This facilitated crystallization of ternary substrate complexes of both the wild-type and Y271A mutant enzymes. Crystallographic structures of Y271A-and wild type-substrate complexes indicated that rCTP is well accommodated in the active site but that O2 of rCTP and the carbonyl oxygen of Tyr-271 or Ala-271 are unusually close (ϳ2.5 and 2.6 Å , respectively). Structure-based modeling indicates that the local energetic cost of positioning these closely spaced oxygens is ϳ2.2 kcal/mol for the wild-type enzyme. Because the side chain of Tyr-271 also hydrogen bonds with the primer terminus, loss of this interaction affects its catalytic positioning. Our results support a model where DNA polymerase ␤ utilizes two strategies, steric and geometric, with a single protein residue to deter ribonucleotide insertion.

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 deoxycounterparts. 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 ␤ to utilize nucleotides with modified sugars. DNA polymerase ␤ 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.To maintain faithful DNA synthesis, DNA polymerases have evolved to select a dNTP from a pool of structurally similar molecules that preserve Watson-Crick base pairing. This is facilitated by geometric constraints (size, shape, and hydrogen bonding potential) imposed by the template strand, primer terminus, and polymerase. Although the fidelity of base substitution errors, and their correction, has been extensively studied, the fidelity of sugar discrimination has received much less attention. It is well recognized that the dNTP pool imbalances influence DNA polymerase fidelity. In this context, cellular rNTP levels are far greater than their dNTP counterparts (1, 2). To prevent significant levels of RNA synthesis during replication and repair, DNA polymerases must inherently discriminate against nucleotides with a ribose sugar (i.e. possessing a 2Ј-OH) and select 2Ј-deoxyribose triphosphates. Previous studies with A-and B-family polymerases have found significant effects on nucleotide incorporation as a result of modifying the deoxyribose ring (3-8). DNA polymerases insert ribonucleotides with a much lower efficiency than deoxynucleotides with the same base due to a slower rate of insertion and weaker binding.DNA polymerase (pol) 2 ␤, an X-family member that also includes pol , pol , and terminal deoxyribonucleotidyltransferase (TdT), has been well characterized kinetically, structurally, and biochemically (9) making it a model DNA polymerase to probe sugar specificity. DNA polymerase ␤ is a critical ...

A key set of reactions for the initiation of new DNA strands during herpes simplex virus-1 replication consists of the primase-catalyzed synthesis of short RNA primers followed by polymerase-catalyzed DNA synthesis (i.e. primase-coupled polymerase activity). Herpes primase (UL5-UL52-UL8) synthesizes products from 2 to ϳ13 nucleotides long. However, the herpes polymerase (UL30 or UL30-UL42) only elongates those at least 8 nucleotides long. Surprisingly, coupled activity was remarkably inefficient, even considering only those primers at least 8 nucleotides long, and herpes polymerase typically elongated <2% of the primase-synthesized primers. Of those primers elongated, only 4 -26% of the primers were passed directly from the primase to the polymerase (UL30-UL42) without dissociating into solution. Comparing RNA primer-templates and DNA primertemplates of identical sequence showed that herpes polymerase greatly preferred to elongate the DNA primer by 650 -26,000-fold, thus accounting for the extremely low efficiency with which herpes polymerase elongated primase-synthesized primers. Curiously, one of the DNA polymerases of the host cell, polymerase ␣ (p70-p180 or p49-p58-p70-p180 complex), extended herpes primase-synthesized RNA primers much more efficiently than the viral polymerase, raising the possibility that the viral polymerase may not be the only one involved in herpes DNA replication.Herpes simplex virus 1 (HSV-1) 2 encodes seven proteins essential for replicating its double-stranded DNA genome; five of these encode the heterotrimeric helicase-primase (UL5-UL52-UL8 gene products) and the heterodimeric polymerase (UL30-UL42 gene products) (1, 2). The helicase-primase unwinds the DNA at the replication fork and generates singlestranded DNA for both leading and lagging strand synthesis. Primase synthesizes short RNA primers on the lagging strand that the polymerase presumably elongates using dNTPs (i.e. primase-coupled polymerase activity). These two protein complexes are thought to replicate the viral genome on both the leading and lagging strands (1, 2).Previous studies have focused on the helicase-primase and polymerase separately. The helicase-primase contains three subunits, UL5, UL52, and UL8 in a 1:1:1 ratio (3-5). The UL5 subunit has helicase-like motifs and the UL52 subunit has primase-like motifs, yet the minimal active complex that demonstrates either helicase or primase activities contains both UL5 and UL52 (6, 7). Although the UL8 subunit has no known catalytic activity, several functions have been proposed, including enhancing helicase and primase activities, enhancing primer synthesis on ICP8 (the HSV-1 single-stranded binding protein)-coated DNA strands, and facilitating formation of the replisome (8 -12). Although primase will synthesize short (2-3 nucleotides long) primers on a variety of template sequences, synthesis of longer primers up to 13 nucleotides long requires the template sequence, 3Ј-deoxyguanidine-pyrimidine-pyrimidine-5Ј (13). Primase initiates synthesis at the first pyrimidine...