Vashishtha Ashwani Kumar, Kuchta Robert D
Department of Chemistry and Biochemistry, University of Colorado , Boulder, Colorado 80309-0215, United States.
Biochemistry. 2016 Feb 23;55(7):1168-77. doi: 10.1021/acs.biochem.6b00065. Epub 2016 Feb 11.
We examined the impact of two clinically approved anti-herpes drugs, acyclovir and Forscarnet (phosphonoformate), on the exonuclease activity of the herpes simplex virus-1 DNA polymerase, UL30. Acyclovir triphosphate and Foscarnet, along with the closely related phosphonoacetic acid, did not affect exonuclease activity on single-stranded DNA. Furthermore, blocking the polymerase active site due to either binding of Foscarnet or phosphonoacetic acid to the E-DNA complex or polymerization of acyclovir onto the DNA also had a minimal effect on exonuclease activity. The inability of the exonuclease to excise acyclovir from the primer 3'-terminus results from the altered sugar structure directly impeding phosphodiester bond hydrolysis as opposed to inhibiting binding, unwinding of the DNA by the exonuclease, or transfer of the DNA from the polymerase to the exonuclease. Removing the 3'-hydroxyl or the 2'-carbon from the nucleotide at the 3'-terminus of the primer strongly inhibited exonuclease activity, although addition of a 2'-hydroxyl did not affect exonuclease activity. The biological consequences of these results are twofold. First, the ability of acyclovir and Foscarnet to block dNTP polymerization without impacting exonuclease activity raises the possibility that their effects on herpes replication may involve both direct inhibition of dNTP polymerization and exonuclease-mediated destruction of herpes DNA. Second, the ability of the exonuclease to rapidly remove a ribonucleotide at the primer 3'-terminus in combination with the polymerase not efficiently adding dNTPs onto this primer provides a novel mechanism by which the herpes replication machinery can prevent incorporation of ribonucleotides into newly synthesized DNA.
我们研究了两种临床批准的抗疱疹药物阿昔洛韦和膦甲酸钠对单纯疱疹病毒1型DNA聚合酶UL30的核酸外切酶活性的影响。三磷酸阿昔洛韦、膦甲酸钠以及密切相关的膦乙酸，均不影响对单链DNA的核酸外切酶活性。此外，由于膦甲酸钠或膦乙酸与E-DNA复合物结合或阿昔洛韦在DNA上聚合而导致的聚合酶活性位点阻断，对核酸外切酶活性的影响也很小。核酸外切酶无法从引物3'-末端切除阿昔洛韦，是由于糖结构改变直接阻碍了磷酸二酯键的水解，而不是抑制结合、核酸外切酶使DNA解旋或DNA从聚合酶转移到核酸外切酶。从引物3'-末端的核苷酸上去除3'-羟基或2'-碳会强烈抑制核酸外切酶活性，不过添加2'-羟基并不影响核酸外切酶活性。这些结果的生物学意义有两个方面。首先，阿昔洛韦和膦甲酸钠能够阻断dNTP聚合而不影响核酸外切酶活性，这增加了它们对疱疹病毒复制的影响可能既涉及直接抑制dNTP聚合又涉及核酸外切酶介导的疱疹病毒DNA破坏的可能性。其次，核酸外切酶能够快速从引物3'-末端去除核糖核苷酸，再加上聚合酶不能有效地将dNTP添加到该引物上，这提供了一种新机制，疱疹病毒复制机制可借此防止核糖核苷酸掺入新合成的DNA中。
