Insertion and extension of acyclic, dideoxy, and ara nucleotides by herpesviridae, human alpha and human beta polymerases. A unique inhibition mechanism for 9-(1,3-dihydroxy-2-propoxymethyl)guanine triphosphate.

R Reid,M D Topal,E S Huang,E C Mar

doi:10.1016/s0021-9258(18)69010-6

Abstract

The ability of human alpha and beta 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-propoxymethyl)guanine (dhpG) and 9-(2-hydroxyethoxymethyl)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, 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 greater than (acyGTP and araNTP) greater than ddGTP, whereas by human alpha polymerase it was araATP greater than ddGTP much greater than (acyGTP and dhpGTP) and by human beta polymerase it was (araATP and ddGTP) much greater than (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 alpha and beta 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 alpha DNA polymerase, which is acyGTP resistant, was found to vary by one amino acid from the amino 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.

Highlights

From the Lineberger Cancer Research Center and the Departments of $Biochemistry, §Medicine, and
Two megalovirus (HCMV) DNA polymerases to insert and of the most effectivecompounds of this group have in common extend several nucleotideanalogs has been investi- incompleteness of the sugar ring: [9-(2-hydroxyethoxygated using a variation of Sanger-Coulson DNA se- methy1)guaninel-triphosphate [1,2,3,4,5]
In thispaper we examine some araNTPs; second, insertionJextensionbehaviorwith effects of nucleotide sugar modification on DNA polymerase dhpGTP; and third, the relative preference for inser- insertion and elongation behavior

Summary

None ddG ocyG

Polymerase Inhibition by Nucleotide Sugar Analogs column. On DEAE-cellulose the HSV-2-induced DNA polymerase, host cellular a polymerase, and DNase copurified a t 0.08 to 0.15 M. For all the DNA polymerases, to be the amount of enzyme required to incorporate 1 nmol of nucleotide/h at 37 "C. One mlof indicated polymerase (0.04 units as defined above of polymerase a,0.08 units of polymerase 8, 0.00076 units ofCMV polymerase, 0.002 units of HSV-2 polymerase), in astorage buffer containing a t least 200 pg/ml of nuclease-free bovine serum albumin, was added to each reaction and the reactions were incubated a t 37 "C for 15 min a t which time reactions were stopped by addition of 5 pl of stop mixture Tabulated integrations of bands werewell within the range found in Ref. 24 to give linear response

RESULTS

Human a

AAA ins i i n nA a A A

HCMV pol

DISCUSSION

EBV HCMV