Benzo[c]phenanthrene Adducts and Nogalamycin Inhibit DNA Transesterification by Vaccinia Topoisomerase

Abstract

Vaccinia DNA topoisomerase forms a covalent DNA-(3-phosphotyrosyl)-enzyme intermediate at a specific target site 5-C ؉5 C ؉4 C ؉3 T ؉2 T ؉1 p2N ؊1 in duplex DNA. Here we study the effects of position-specific DNA intercalators on the rate and extent of single-turnover DNA transesterification. Chiral C-1 R and S trans-opened 3,4-diol 1,2-epoxide adducts of benzo[c]phenanthrene (BcPh) were introduced at single N 2 -deoxyguanosine and N 6 -deoxyadenosine positions within the 3-sequence of the nonscissile DNA strand. Transesterification was unaffected by BcPh intercalation between the ؉6 and ؉5 base pairs, slowed 4-fold by intercalation between the ؉5 and ؉4 base pairs, and virtually abolished by BcPh intercalation between the ؉4 and ؉3 base pairs and the ؉3 and ؉2 base pairs. Intercalation between the ؉2 and ؉1 base pairs by the ؉2R BcPh dA adduct abolished transesterification, whereas the overlapping ؉1S BcPh dA adduct slowed the rate of transesterification by a factor of 2700, with little effect upon the extent of the reaction. Intercalation at the scissile phosphodiester (between the ؉1 and ؊1 base pairs) slowed transesterification by a factor of 450. BcPh intercalation between the ؊1 and ؊2 base pairs slowed cleavage by two orders of magnitude, but intercalation between the ؊2 and ؊3 base pairs had little effect. The anthracycline drug nogalamycin, a noncovalent intercalator with preference for 5-TG dinucleotides, inhibited the single-turnover DNA cleavage reaction of vaccinia topoisomerase with an IC 50 of 0.7 M. Nogalamycin was most effective when the drug was preincubated with DNA and when the cleavage target site was 5-CCCTT2G instead of 5-CCCTT2A. These findings demarcate upstream and downstream boundaries of the functional interface of vaccinia topoisomerase with its DNA target site.Poxvirus DNA topoisomerase I is important for virus replication (1) and a potential target for drug therapy of smallpox, in light of its unique DNA recognition specificity, compact structure, and distinctive pharmacological sensitivities compared with human topoisomerase I (2-6). Poxvirus topoisomerases are exemplary type IB family members; they cleave and rejoin one strand of the DNA duplex through a transient DNA-(3Ј-phosphotyrosyl)-enzyme intermediate. Vaccinia topoisomerase cleaves duplex DNA at a pentapyrimidine target sequence, 5Ј-(T/C)CCTTp2 (3). (The Tp2 nucleotide is defined as the ϩ1 nucleotide.) Topoisomerases encoded by other genera of poxviruses recognize the same DNA target sequence (6 -10). Available structural and biochemical studies suggest that the assembly of a catalytically competent topoisomerase active site is triggered by recognition of the DNA target (11,12).Early studies using nuclease footprinting, modification interference, modification protection, analog substitution, and UV crosslinking techniques suggested that vaccinia topoisomerase makes contact with several nucleotide bases and the sugar-phosphate backbone of DNA within and immediately flanking the CCCTT element (13)(14)(15)(16)(17)(18)(19). Re...

Individual Nucleotide Bases, Not Base Pairs, Are Critical for Triggering Site-specific DNA Cleavage by Vaccinia Topoisomerase

“…Role of the Ϫ1 Base in DNA Cleavage-Vaccinia topoisomerase displays no preference for a particular base pair immediately 3Ј of the scissile phosphodiester (1,18). Here we found that an abasic lesion at Ϫ1 on the scissile strand slowed k cl by a factor of 350, whereas a Ϫ1 abasic lesion on the nonscissile strand had no effect at all.…”

“…Recent studies have focused on delineating the features of the DNA interface that affect the kinetics of transesterification. For example, positionspecific covalent polycyclic aromatic hydrocarbon diol epoxide-DNA adducts have been exploited to probe the minor groove interface (16) and the effects of intercalation at all of the dinucleotides steps spanning the target site (17,18). The aromatic hydrocarbon adduct studies delineated the margins of the functional DNA interface at atomic resolution but did not reveal the nature of the DNA contacts within the essential zone of DNA.…”

“…Whereas the affinity for the target site, the rate of cleavage, and the cleavage equilibrium constant (K cl ) are affected by the nucleotide sequence context surrounding the 5Ј-(C/T)CCTT target site (1,15) in ways that are not well understood in structural terms, the dominant factor triggering the DNA incision reaction is the pentamer 5Ј-CCCTT/3Ј-GGGAA. We have begun to systematically address the features of the individual bases that affect the kinetics of DNA cleavage via position-specific base modifications entailing relatively small additions to, or subtractions from, the standard base structures (11,17), as well as modification by more bulky adducts (16,18). The addition of new substituents to the purine and pyrimidine rings provides a means of mapping functionally relevant sites of protein-DNA contact.…”

Nonpolar Nucleobase Analogs Illuminate Requirements for Site-specific DNA Cleavage by Vaccinia Topoisomerase

“…Position-specific base modifications have been used to systematically address the features of the individual target site bases that affect the kinetics of DNA transesterification (13)(14)(15)(16)(17). The sensitivity of this method depends on the degree to which the particular modification alters the size, shape, stacking, and hydrogen-bonding potential of the base or base pair.…”

Major Groove Interactions of Vaccinia Topo I Provide Specificity by Optimally Positioning the Covalent Phosphotyrosine Linkage

“…For instance, site-specific modification with benzo-[c]-phenanthrene at N2 and N6 of G can reduce the cleavage rate by a factor of 1000 (6), and site-specific modification of the exocylic amino groups of the +1 and +2 adenines with benzo-[a]-pyrene decreased the rate of cleavage by approximately 10 3 -fold (15). The rate effects on strand cleavage arising from the more subtle major groove modifications of 8-oxoguanine, 8-oxoadenine and 2-aminopurine have also been explored, where deleterious effects as large as 35-fold at the +3G were observed (15).…”