title: Polyadenylation inhibition by the triphosphates of deoxyadenosine analogues.
abstract: The nucleotide substrate specificity of yeast poly(A) polymerase (yPAP) was examined with various ATP analogues of clinical relevance. The triphosphate derivatives of cladribine (2-Cl-dATP), clofarabine (Cl-F-ara-ATP), fludarabine (F-ara-ATP), and related derivatives were incubated with yPAP and 32P-radiolabeled RNA oligonucleotide primers in the absence of ATP to assay polyadenylation. While 2-Cl-ATP resulted in primer elongation, ara-ATP and F-ara-ATP were poor substrates for yPAP. In contrast, the triphosphate derivatives of cladribine (2-Cl-dATP), clofarabine (Cl-F-ara-ATP) and its corresponding deoxyribose derivative (Cl-F-dATP) were substrates and caused chain termination in the absence of ATP. We further investigated whether analogue incorporation at the 3'-terminus of RNA primers negatively impacts polyadenylation with ATP by generating RNA oligonucleotides containing either a terminal clofarabine, Cl-F-dAdo, or cladribine residue. Incorporation of any of these analogs blocks the ability of yPAP to extend RNA past the analogue site, impeding the addition of a poly(A)-tail. To determine whether modified ATP analogues exhibit a concentration-dependent effect on polyadenylation, poly(A)-tail synthesis by yPAP with modified ATP analogues in combination with a constant level of ATP was also examined. With all the ATP analogues assayed in these studies, there was a significant reduction in poly(A)-tail length with increasing amounts of analogue triphosphate. Taken together, our results suggest that polyadenylation inhibition may be a component in the mechanism of action of adenosine analogues.
authors.affiliation: Department of Experimental Therapeutics, UT MD Anderson Cancer Center, Houston, TX 77030, USA.
authors.forename: Lisa S
authors.initials: LS
authors.lastname: Chen
authors.forename: William
authors.initials: W
authors.lastname: Plunkett
authors.forename: Varsha
authors.initials: V
authors.lastname: Gandhi
mesh_terms: D000227:Adenine Nucleotides / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives*; D000970:Antineoplastic Agents / Q000737:chemistry*; D001087:Arabinonucleosides / Q000737:chemistry; D017338:Cladribine / Q000737:chemistry; D000077866:Clofarabine; D003838:Deoxyadenine Nucleotides / Q000737:chemistry* / Q000378:metabolism; D003839:Deoxyadenosines / Q000737:chemistry; D026723:Polyadenylation; D011062:Polynucleotide Adenylyltransferase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D000227:Adenine Nucleotides; D000970:Antineoplastic Agents; D001087:Arabinonucleosides; D003838:Deoxyadenine Nucleotides; D003839:Deoxyadenosines; D017338:Cladribine; D000077866:Clofarabine; D000255:Adenosine Triphosphate; D011062:Polynucleotide Adenylyltransferase