Insertion and Extension of Acyclic, Dideoxy, and Ara Nucleotides by Herpesviridae, Human cu and Human Polymerases A UNIQUE INHIBITION MECHANISM FOR 9-(1,3-DIHYDROXY-2-PROPOXYMETHYL)GUANINE TRIPHOSPHATE*

The ability of human a and B DNA polymerases and herpes simplex virus type 2 (HSV-2) and human cyto-megalovirus (HCMV) DNA polymerases to insert and extend several nucleotide analogs has been investi- gated using a variation of Sanger-Coulson DNA sequencing technology. The analogs included the tri- phosphates of two antiviral nucleosides with incom-plete sugar rings: methy1)guanine or acyclovir), as well as dideoxy and arabinosyl nucleoside triphosphates. Three pairs of contrasting behaviors were found, each pair distinguishing the two human polymerases from the two viral ones: first, extension behavior with araNTPs; second, insertionJextension behavior with dhpGTP; and third, the relative preference for insertion of ddGTP uersus acyGTP. The relative level of insertion of the nucleotide analogs by HCMV and HSV-2 DNA polymerases was dhpGTP > (acyGTP and araNTP) > ddGTP, whereas by human a polymerase

The ability of human a and B DNA polymerases and herpes simplex virus type 2 (HSV-2) and human cytomegalovirus (HCMV) DNA polymerases to insert and extend several nucleotide analogs has been investigated using a variation of Sanger-Coulson DNA sequencing technology. The analogs included the triphosphates of two antiviral nucleosides with incomplete sugar rings: 9-( 1,3-dihydroxy-2-propoxy-methy1)guanine (dhpG) and 9-(2-hydroxyethoxy-methy1)guanine (acyG or acyclovir), as well as dideoxy and arabinosyl nucleoside triphosphates.
Three pairs of contrasting behaviors were found, each pair distinguishing the two human polymerases from the two viral ones: first, extension behavior with araNTPs; second, insertionJextension behavior with dhpGTP; and third, the relative preference for insertion of ddGTP uersus acyGTP. The relative level of insertion of the nucleotide analogs by HCMV and HSV-2 DNA polymerases was dhpGTP > (acyGTP and araNTP) > ddGTP, whereas by human a polymerase it was araATP > ddGTP > > (acyGTP and dhpGTP) and by human B polymerase it was (araATP and ddGTP) >> (acyGTP and dhpGTP).
Evidence is presented for three mechanisms of inhibition by extendible nucleotides (of dhp and ara types) exhibiting frequent internalization: araATP acted as a simple pseudoterminator of a and B polymerases, but was easily extended past singlet sites by Herpesviridae polymerases and only stalled at sites requiring two or more araATP insertions in a row. Herpesviridae polymerases stalled after adding dhpGMP and one additional nucleotide, suggesting that polymerase translocation problems may be a factor in polymerase inhibition by modified sugar nucleotide analogs.
The amino acid sequence of the human a DNA polymerase, which is acyGTP resistant, was found to vary by one amino acid from the amino acid sequences of the Herpesviridae polymerases in a region of significant similarity and probable functional homology. Amino acid differences at that same site differentiate acyclovir-resistant HSV-1 mutants from the acyclovirsensitive HSV-1 wild type.
Several nucleotide analogs based on modification of the * This work was supported by United States Public Health Service Grants GM24798 and CA28632 (to M. D. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
11 Scholar of the Leukemia Society of America. To whom correspondence should be addressed.
The detailed mechanisms of polymerase inhibition by these (and other) extensively investigated nucleotide analogs are still only partially elucidated. In this paper we examine some effects of nucleotide sugar modification on DNA polymerase insertion and elongation behavior. The modified-sugar nucleotide analogs investigated were of the following four types, where N refers to some normal base: 2',3'-dideoxyNTP (ddNTP), acyNTP, dhpNTP, and 9-P-D-arabinofuranosyl-NTP (araNTP). Two types can be not only inserted but also extended by various polymerases (ara and dhp nucleotides); they have nevertheless the potential to dramatically decrease the overall rate of polymerization (8). The other two force termination of DNA replication once incorporated (dd and acy nucleotides, which lack a 3'-OH).
The polymerases used were human a, human P, and viral polymerases from two main branches of the Herpesviridae, type a (HSV-2) and type P (HCMV). These polymerases are known to vary in their sensitivities to inhibition by the four analog types. For example, polymerase p has been shown to be more sensitive to dideoxy nucleotides and less sensitive to ara nucleotides than are a and HSV-1 polymerases (9-11) (see also Ref. 12 for HSV-2 and ara), whereas the Herpesviridae polymerases are strikingly more sensitive to the acyclic analogs (acy and dhp) (see Ref. 8 for review).
Inhibition by each of the nonterminating analogs has been seen as paradoxical. It has been proposed that ara nucleotides act effectively as chain terminators (13). This was, however, followed by work showing not only that ara nucleotides could be polymerized into closed circular DNA, but that under normal conditions of inhibition most ara residues were to be found internally rather than terminally in the primer strand (14). Similarly, a study in vitro of dhpGTP inhibition of polymerases of the herpes family (4) found a minimum of 50% of incorporated dhpG to be extended in the presence of other triphosphates, this despite the fact that under similar conditions dhpG-terminated template-primers are strong competitive inhibitors (7). For some of the polymerases involved, these results are complicated by the presence of 3'-5"exonuclease activity (15).
For the terminating analogs, dd and acy nucleotides (ie. the sugar analogs that cannot be extended for lack of a 3'-OH), at least four separate mechanisms of inhibition have been discussed (a) competitive inhibition by the nucleotide (as above, tight binding but slow insertion); (b) chain termination, depleting the primer pool; ( c ) competitive inhibition by the analog-terminated primer (4, 7, 16) (which can be strongly affected by the concentration of the required next nucleotide (16)); and ( d ) suicide inhibition, in which the polymerase is inactivated after an encounter with templateprimer and nucleotide analog (5,17). In particular, it is unclear whether the kinetic behavior that led to mechanism d is actually due to formation of a covalent bond. We provide evidence against this bond and suggest instead an alternate form of mechanism c, wherein the template-primer binds in an alternative manner to that for which kinetic behavior is being measured in normal competitive inhibition protocols.
We present a DNA sequencing approach that provides a direct assay for the relative abilities of different polymerases to extend specific DNA primers by inserting one or a few nucleotides followed by termination or stalling. In particular, it allows a search for site-specific stalling influenced, for example, by nearby internalized analogs. Evidence is presented showing that for a given inhibitor the mechanism of inhibition may vary for different polymerases (e.g. CY versus Herpesviridae polymerases with araNTP).
Three pairs of contrasting behaviors were found, each pair distinguishing the two human polymerases from the two viral ones: first, extension behavior with araNTPs; second, insertion/extension behavior with dhpGTP; and third, the relative preference for insertion of ddGTP versus acyGTP. The relative level of insertion of the nucleotide analogs by HCMV and HSV-2 DNA polymerases was dhpGTP > (acyGTP and araNTP) > ddGTP, whereas by human CY polymerase it was araATP > ddGTP >> (acyGTP and dhpGTP) and by human fi polymerase it was (araATP and ddGTP) >> (acyGTP and dhpGTP).
Evidence is presented for three mechanisms of inhibition by extendible nucleotides (of dhp and ara types) exhibiting frequent internalization: araATP acted as a simple pseudoterminator of CY and fi polymerases, but was easily extended past singlet sites by Herpesviridae polymerases and only stalled at sites requiring two or more araATP insertions in a row. Herpesviridae polymerases stalled after adding dhpGMP and one additional nucleotide, suggesting that polymerase translocation problems may be a factor in polymerase inhibition by modified sugar nucleotide analogs.
The relative insertion behaviors of nucleotides with all four types of sugar modification are compared to the published inhibition behaviors as measured by KI.
The amino acid sequence of the human CY DNA polymerase, which is acyGTP-resistant, was found to vary by one amino acid from the amino acid sequences of the Herpesviridae polymerases in a region of significant similarity and probable functional homology. Amino acid differences at that same site differentiate acyclovir-resistant HSV-1 mutants from the acyclovir-sensitive HSV-1 wild type. HSV-2-induced DNA polymerase was purified by successive chromatography on DEAE-cellulose and phosphocellulose, as previously reported (20), and further purified on a single-stranded DNA-agarose One unit is defined, for all the DNA polymerases, to be the amount of enzyme required to incorporate 1 nmol of nucleotide/h at 37 "C.

Nucleotides
The specific activities of the HCMV, HSV-2, a and 8 enzymes were, respectively, 3.2,4.2,4.4, and 2.7 units/pg of protein, when measured under assay conditions as described in Refs. 18 and 19.
Sequencing Reactions-Minus reactions were performed essentially as described by Sanger and Coulson (21) with the modifications of template-primer concentrations at one-tenth normal and the addition of bovine serum albumin to stabilize polymerases. The templateprimer was synthetic 12-mer equivalent to a portion of the ampicillinase gene of pBR322 (pCAATAAACCAGC, positions 3467 to 3478) annealed to fl-R208 single-stranded DNA, which is an fl/pBR322 chimera containing the negative strand of pBR322 (22). The replicating daughter-strand sequence shown agrees with the published sequence for pBR322 (23). The initial reaction mixture contained 0.13 pg of template-primer, 0.05 mM dATP, dTTP, and dGTP, 10 pCi of [cY-~*P]~CTP (400-800 Ci/mmol), 25 mM Tris-HC1 (pH 7.5), 10 mM MgCI,, 1 mM 8-mercaptoethanol, and 50 mM NaCl. Ten units (one unit being defined, as for all the polymerases, to be 1 nmol of nucleotide incorporated per h a t 37 "C) of Klenow Escherichia coli polymerase I (Boehringer Mannheim or Bethesda Research Laboratories) in 1 pl of 50 mM KPi, pH 7.4, 0.25 mM dithiothreitol, 50% glycerol were added and the reaction was incubated on ice. At 5, 10, and 15 min, 6 p l , 12 p l , and the remainder of the mixture, respectively, were removed (volumes and times chosen to give a preferred spectrum of primer lengths) and the reaction was stopped by addition to chloroform/phenol/H20/isoamyl alcohol (23:2525:0.5). The aqueous phase was removed, extracted with ether, applied to a 1-ml column of Sephadex G-100, and eluted with 1 mM Tris-HC1 (pH 7.5), 0.5 mM EDTA. The leading radioactive peak containing 32P-labeled DNA was collected, lyophilized to dryness, and resuspended in 50 pl of 50 mM Tris-HCI, pH 8.0 at 0 "C, 10 mM MgCI,, 1 mM dithiothreitol. Two pl of the resuspension were added to 2 pl of the appropriate minus mixture (25 p~  None ide). The stopped reactions were heated to 100 "C for 3 min, quickly cooled in an ice/water bath, and loaded onto an 8% polyacrylamide gel (34 cm X 0.4 mm) containing gel buffer (0.09 M Tris acetate, 0.09 M boric acid, 2.5 mM Na2EDTA, pH 8.3) plus 7 M urea. The gel was electrophoresed at 1500-1700 V until the bromphenol blue reached the bottom of the gel (-2 h). An autoradiogram was obtained at -20 "C. Densitometry-Gel autoradiograms were scanned on a Bio-Rad 1650 densitometer (Hoefer). Scans were made at 6.5 cm/min and were analyzed using the Appligration system of Dynamic Systems Corp. Tabulated integrations of bands were well within the range found in Ref. 24 to give linear response.

RESULTS
DNA Sequencing Assay-A DNA sequencing approach based on the Sanger method (21,25,26) was used to directly study nucleotide analog insertion. The template-primer concentration was often kept low in our approach, compared to normal DNA sequencing, to enable use of polymerase preparations (human a and HCMV) at activities orders of magnitude below those (e.g. of Klenow DNA polymerase I) normally used for DNA sequencing. The primer/polymerase ratio was still high enough for a and HCMV polymerases so that almost all active primers (that is, primers which were extended by at least one nucleotide) were expected to have only one encounter with a polymerase (one cycle of processive synthesis).
The sequencing approach is illustrated using ddGTP and acyGTP with human /3 DNA polymerase (Fig. 1). A spectrum of different length DNA primers (shown in the "no-polymerase" lane) was offered to / 3 polymerase for extension, either in the absence of dGTP (the second lane) or with dGTP replaced by ddGTP or acyGTP (the next two lanes). In each case, the remaining three normal nucleoside triphosphates were present. In the absence of dGTP, primers were extended to the sites before G sites in the growing-strand sequence. The polymerase could extend only a fraction of the primers represented in each band, evidenced by intensity remaining at original band positions. The strongest original bands correspond to high-frequency primers (e.g. position G3497) produced under the C-limiting conditions used when the primer mixture was made (see "Materials and Methods"). Added ddGTP was efficiently inserted in place of dGTP at many sites, terminating replication a t those sites. One particular sequencing gel area (shown by brackets in Fig. 1 Fig. 2; a lighter exposure was used for densitometry. a, an overlay of scans of two lanes showing Q polymerase use of araATP. One scan is of the "-A" lane, which shows the results of a 1-h incubation with human a polymerase and 25 p~ each of three normal triphosphates (dGTP, dCTP, and dTTP); the other is the same, except that araATP is present at 50 p~. The peak, which is more intense in the minus-adenosine scan (C3498). is shaded where it exceeds the equivalent peak of the scan of the araATP lane; the peak more intense in the araATP lane scan (where araATP has inserted at the position of A3499) is hatched. b, an overlay of scans of two lanes showing HCMV polymerase use of araATP. As in a, the lanes show the results of incubating the polymerase for 1 h with reaction mixtures that include three normal triphosphates at 25 pM (dGTP, dCTP, and d l T P ) , but either, first, no analog for dATP, or second, 50 p~ araATP. Again, the araATP-induced decrease in intensity (at C3498) is shown by shading, and the increases are hatched. In the region shown, most strands used primers a t G3497; above certain peaks are shown the inferred extensions. Note that each insertion of an ara-nucleotide retards the migration rate (which is left to right) slightly more than insertion of a deoxy-nucleotide. Nucleotide Analog Insertion and Extension-The abilities of four DNA polymerases (polymerase a, polymerase j3, HSV-2 polymerase, and HCMV polymerase) to insert acyGTP and ddGTP and to insert and possibly extend dhpGTP, araATP, and araGTP were compared (Figs. 2-5 dhpGTP. Conditions were as described in legend to Fig. 2. 6, densitometric scans of selected lanes of the autoradiogram of the gel; a lighter exposure was used for densitometry. An overlay of scans of two lanes shows HCMV polymerase use of dhpGTP. One scan is of the " 4 " lane, which shows the results of a 1-h incubation with human HCMV polymerase and 25 p~ each of three normal triphosphates (dATP, dCTP, and dTTP); the other is the same, except that dhpGTP is present at 50 p~. The peak that is more intense in the first scan (A3499) is shaded where it exceeds the equivalent peak of the scan of the dhpGTP lane; the increased intensities in the second scan are hatched.

E E G A A A ins i i n A n a A A A* A* A*
F begins) corresponds to the band at G3497; only the region immediately above it is shown, but the behaviors described are repeated at other sites, as exemplified in Fig. 1. The a and HCMV polymerases were at low specific activity, so long exposure was used to make the bands in the C3498 to A3502 region more easily visible (Fig. 2). For each of 01 and HCMV polymerases, the peaks at A3499 in the -G lanes, which serve as a measure of the original C3497 primers that were extended, are much less than the residual peaks at G3497.
Qualitatively, band appearance at G3500 (Figs. 2 and 5) demonstrated, for example, that ddGTP was incorporated by all polymerases studied, as indicated schematically at the bottom of the figures. For quantitative comparisons, less exposed autoradiograms of the gels were analyzed by densitometry (Figs. 3 and 4; Tables I and 11). Scans of the araA and dhpG lanes are shown in the figures; the ddG and acyG lanes are simpler, because insertion implies termination, whereas for araA and dhpG both insertion and extension must be followed.
Ara Nucleotide Insertion and Extension-The behavior of human 01 polymerase in minus reactions without analog was to add one dCMP subunit to a rather small fraction of the primers ending with G3497 to produce a shoulder at C3498 (Fig. 3). In the parallel reaction with 50 mM araATP, the area of the shoulder was decreased by more than half (the shaded region), and a new peak appeared (the hatched peak) near (but slightly to the left of) the A3499 site (Fig. 3). The fraction   of G3497 primers that inserted (at least) the dCMP at C3498 (which we will call the "active" primers) should be the same in the two reactions, so the decrease in the C3498 shoulder is a measure of the active primers that are further extended by araATP insertion. Although over half of the active primers (about 70%, by densitometry; see Table I) extended the C3498 strands with araATP, there was no significant further extension seen either at G3500 or at the next A site, A3501.

Fraction extended by polymerase (pol) indicated from C3498 to
Insertion of an ara nucleotide retarded migration slightly more than insertion of deoxy, dideoxy, or acy nucleotides with the same base (araA and araG lanes in Figs. 2 and 5). This shifts bands slightly higher on the gels, and, equivalently, slightly to the le& on the densitometric scans, thus aiding in the visual and densitometric identification of increased bands involving ara nucleotides.
HCMV polymerase in the presence of araATP (Fig. 3) extended well over half of the active primers, as shown by the decrease in the C3498 shoulder from the "-A" lane scan to the "-A plus araA" scan. More than half of these extended primers have been further extended by (presumably) adding G at the G3500 site and then (usually) a second araA at the A3501 site. There, however, the enzyme apparently pauses, as no insertion corresponding to A3502 can be detected (it should run between the normal A3502 and G3503 peaks). This represents a significant blockage, since about half of the active G3497 primers (as measured by the sum of the hatched areas in Fig. 3, plus the residue remaining at C3498) are extended to the A3501 nucleotide position ( Table I). As in the a case, the large proportion of unextended G3497 primers implies that the extensions shown represent single encounters of polymerase with template-primer (single processivity events).
Inhibition of HCMV and HSV-2 DNA Polymerases by dhpGTP-The experiments with dhpGTP provided a surprising result. When HCMV or HSV-2 DNA polymerases were used, a band appeared at a level two nucleotides above the -G band (A3499) (see Figs. 4 and 5). This could have been due to insertion of dhpGTP alone, if the inserted nucleotide retarded primer electrophoretic mobility twice as much as inserted dGTP or acyGTP alone. To test this possibility, dGTP, ddGTP, araGTP, and dhpGTP were all added to the end of a primer by avian myeloblastosis virus reverse transcriptase in the absence of dNTP needed for additional extension. Electrophoresis of the products on a denaturing DNA sequencing gel indicated that the greatest retardation occurred in the primer capped by araGMP; no discernible difference was seen between the effect of dGMP and that of dhpGMP. Thus, insertion of dhpGTP at G3500 by HCMV or HSV-2 polymerases allowed insertion of one additional nucleotide (in Fig. 4, this would be dATP) followed by stalling.
The stalling is not absolute, as can be seen by densitometric overlay (Fig. 4). Here the active extensions of the G3497 primer by HCMV polymerases are shown by the peak at A3499 in the scan of the "-G" lane, the result of adding dCMP and then dAMP to the primer. The shaded decrease in the A3499 peak, in the parallel reaction with dhpGTP, measures the insertion of dhpGTP at the G3500 sites, whether further extended or not. The hatched area at A3501 and the smaller one at T3504 show the preferred pause or termination sites for a processive cycle. Each follows a G site; the peak at T3504 shows the degree to which the polymerase can overcome the first block and extend to a second similar one.
Insertion and Termination by ddGTP and acyGTP-A summary of densitometric data from the autoradiograms is given in Table I1 for the terminators ddGTP and acyGTP. Since each is a G analog and a terminator, the active G3497 primers that have been extended as far as A3499, the normal block site under -G conditions, should be partitioned in each case between the A3499 band (if not further extended) and the G3500 band (if the analog has been inserted). The table gives the fraction at each site. p polymerase shows a higher ddGTP incorporation than a or HCMV, but for all three the incorporation is significant (0.6, 0.4, and 0.3, by densitometric estimate (Table I)).
Relative Insertion of Nucleotide Analogs-For each polymerase, the gels also provide a rank ordering of the insertion rates of the nucleotide analogs.
In particular, they show dhpGTP to be a slightly more effective substrate for insertion than is acyGTP for each of the two Herpesviridae polymerases (Table 11), despite the fact that in each case acyGTP has the lower Kl (reviewed in Ref. 8) by a factor of 2.5 to 10. For HCMV, this can be seen by comparing the 50 mM acyGTP lane in Fig. 2 to the 5 and 50 mM lanes in Fig. 4; the 50 mM acyGTP extended about half the available primers, the 5 mM dhpGTP nearly half, and the 50 mM dhpGTP well over half.
For HSV-2 (Fig. 5), the acyGTP lanes show that at 500 nM acyGTP the ratio of the intensity at the G3500 site (insertion of acyGTP) to that at the A3499 site (waiting for extension by dGTP analog) is just over 1:1, while a t 50 nM acyGTP the ratio is much less. For dhpGTP insertion, the ratio of the intensity at A3501 to that at A3499 (Fig. 5 ) show that, at 20 nM dhpGTP, insertion is at least comparable to acyGTP insertion at 50 nM. At 500 nM dhpGTP, enough strands are extended past the A3501 stall point that the next dhpGTPspecific stall point (at T3504, after G3503; compare Fig. 4) becomes the most intense of the extensions (results not shown); the sum of the bands representing insertion of dhpGTP at the G3500 site (plus further extensions) is then significantly greater than the band at A3499 representing failure to insert.
For each of the HCMV and HSV-2 polymerases, the level of insertion could be ordered as follows: dhpGTP > (acyGTP and araNTP) > ddGTP. This order is not the same as the order by inhibition strength as measured by (Kl)-', which is acyGTP > dhpGTP > araNTP > ddNTP (8).
Although the cellular polymerases differ in sensitivity to ddGTP (/3 is well known to be more sensitive to ddNTP inhibition than CY), they each insert ddGTP significantly at concentrations a t which acyGTP insertion is not detectable on the gels. The order for level of insertion is araATP > ddGTP >> (acyGTP and dhpGTP) for a polymerase and (araATP and ddGTP) > > (acyGTP and dhpGTP) for /3 polymerase.

DISCUSSION
For each of the cases (ara and dhp) in which termination is not forced, the mechanistic possibilities considered by previous workers have included competitive inhibition (ie. tight binding but low or slow insertion) and pseudotermination (i.e. slow extension after insertion) (8,27,28). In this work, three types of nonterminating inhibition were seen, exhibited respectively by: (i) Herpesviridae polymerases with dhpGTP, (ii) Herpesviridae polymerases with ara nucleotides (araATP and araGTP), and (iii) a and p polymerases with ara nucleotide (araATP).
AraATP with cy and p acted as a simple pseudoterminator, with slow extension after any insertion. (Although both CY and p exhibit the same mechanism, pseudotermination, with araATP, they are distinguishable from each other by a lower KI for fi than for cy polymerases (9)). Herpesviridae polymerases, however, easily extended ara nucleotides at singlet sites, but stalled when attempting to insert an additional ara nucleotide at runs of two or more consecutive ara sites Or in other ara-rich regions.
In the third type of inhibition, the Herpesviridae polymer-ases were stalled after adding dhpGMP and one additional nucleotide. This suggests that in vivo, and in assays where dhpGTP and following nucleotides are present, most dhpGMP subunits that are incorporated by Herpesviridae polymerases should be internal rather than at termini. This is consistent with studies (4) that found that a t least half of the dhpGMP subunits incorporated into activated calf thymus DNA (by HSV-1 polymerase) were internal. (This amount may be too low: as any phosphatase contamination in the assay (as discussed in Ref. 14) would have lowered the result.) Stalling of polymerization after internalization in the growing strand may explain the apparent contradiction (7, 27) that in certain cases polymerases find it easy to internalize a nucleotide analog, while at the same time total polymerization is strongly inhibited. One mechanism for such stalling would be a delayed fraying of the template-primer; dhpG. C base pairs might initially be held together with the help of the polymerase, but then induce fraying after two polymerase translocations. Perhaps more likely, interaction with the missing portion of the sugar may be required for efficiency in some step in the polymerase's normal sequence of activities, the most obvious possibility being the second translocation after insertion. This model, then, proposes that the polymerase inserts a dhpGMP subunit, translocates to the next site, inserts the appropriate next nucleotide, and then is strongly inhibited (but not totally prevented) from proceeding through the next translocation, that is, that Herpesviridae DNA polymerases depend for normal translocation on a correct interaction with the second sugar from the terminus. In fact, it is possible that this model is more general among polymerases. Since the concentrations used here were not high enough to allow detection of insertion of dhp nucleotides by the cellular polymerases, their extension behavior after insertion of dhpG remains untested.
It might be mentioned that translocation problems may contribute to inhibition mechanisms of terminators. For acyGTP, there may be two separate modes of inhibition by analog-terminated template-primer, the first characterized by KI values in the low nanomolar range (4), and the second by even lower (or nonexistent) off rates, which has led to the suggestion that suicide inhibition might be involved (see Ref. 5 and compare also Ref. 17). The results presented here give evidence against inactivation by formation of a covalent bond between primer and DNA polymerase, since the mobility of the analog-terminated primer in sequencing gels was not disturbed. The sequencing gels show (a) acyGTP attached to primers and (b) the enzyme not attached to primers. A likely explanation for the appearance of suicide inhibition might involve the fact that polymerases bind template-primers in two modes: pretranslocation (after insertion) and post-translocation (or equivalently the initial binding mode). The appearance of suicide inhibition might then be due to binding in the first mode, reached through an insertion event rather than through initial binding, with an inhibition of translocation after insertion being a potential source of slower dissociation.

s important to acyclovir triphosphate sensitivity
The polymerases belong to a superfamily characterized by sequence homology that seems to include all polymerases sensitive to aphidi-Colin. In the consensus sequence, "h" (for "hydrophobic") is used for I, L, V, M, A, G, F, and Y. The A+V and S+N mutations indicated have each been identified (see Ref. 38, in whose terminology this is a portion of Region 11) in acyclovir-resistant HSV-1 mutants. Variation from A and S at these sites is marked with a double underline; variation from the consensus at other sites is marked with a single underline. The partial sequence from human a polymerase was provided by Teresa Wang (Stanford Univ.); also included are five sequences from the herpesvirus family (31-34, 37, 41 -EGMVFDVNSLYPANMYk?RLL -=

Ad 2 P L Y V Y D I S G M Y A S A L ( T -) H P M
some of the characteristic behaviors presented here distinguish members of the superfamily, it is possible to attempt to relate the behaviors to particular areas of high homology and to variations inside those areas. Point mutations affecting inhibition sensitivity have been identified by sequencing in the homologous regions of various members of the superfamily, including the polymerases of HSV-1, HSV-2, and vaccinia virus (38, 39,40). Each of these mutations seems to affect sensitivity to many of a group of inhibitors that includes aphidicolin, pyrophosphate analogs, and nucleotide analogs such as acyGTP (see Ref. 41 for a review of HSV drug resistance). A portion of one of these regions is shown in Table 111. It includes two of the sites implicated in acyGTP sensitivity in HSV-1: A -+ V and S -+ N mutations are from separate HSV-1 acyclovir-resistant mutants, with 19-and 47-fold decrease in sensitivity, respectively; the A V mutation shown is sufficient to induce acyclovir resistance, and the S -+ N mutation is thought to be the more important of two changes found in the other resistant polymerase (see Ref. 38 and see also Ref. 39 for discussion of the same A + V mutation in a different HSV-1 strain). It may be of interest then that in the table the A -+ V site can be seen to differentiate between the Herpesviridae, whether of type a (varicella zoster virus and HSV-l), @ (HCMV), or y (Epstein-Barr virus), and all other members Nucleotide Sugar Analogs of the superfamily. The human a has especially high homology, with a match of 14/15 in a stretch that varies only at the site of the HSV-1 A 4 V mutation; the vaccinia virus (Poxviridae) sequence matches the consensus at 10/13, or 11/13 if the highly homologous F c, Y match is counted, the two clear mismatches being at the two mutant sites.