Polyadenylation inhibition by the triphosphates of deoxyadenosine analogues.
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.
Chen
Lisa S
LS
Department of Experimental Therapeutics, UT MD Anderson Cancer Center, Houston, TX 77030, USA.
Plunkett
William
W
Gandhi
Varsha
V
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
D000227:Adenine Nucleotides; D000970:Antineoplastic Agents; D001087:Arabinonucleosides; D003838:Deoxyadenine Nucleotides; D003839:Deoxyadenosines; D017338:Cladribine; D000077866:Clofarabine; D000255:Adenosine Triphosphate; D011062:Polynucleotide Adenylyltransferase