A human factor that recognizes DNA substituted with 2‐chloroadenine, an antileukemic purine analog

Abstract

2-Chloro-2'-deoxyadenosine (cladribine), an analog of deoxyadenosine, is an important new drug for the treatment of hairy cell leukemia and other forms of adult and pediatric leukemia. By a gel-shift binding assay, we identified an activity in HeLa nuclear extracts that recognizes and binds to oligonucleotides substituted with 2-chloroadenine (ClAde). The activity was specific for ClAde residues because control oligomers did not readily compete out the complex. The binding factor was a monomeric protein that was resistant to inactivation by heating at 45 degrees C but sensitive to heating at 65 degrees C, proteinase K treatment, and 5 mM ZnCl2. This protein, designated ClAde recognition protein (CARP), appeared to be related to a protein that recognized other forms of DNA damage. Gel-shift binding reactions with ultraviolet (UV)-irradiated oligomers revealed a UV-specific protein/DNA complex that had an electrophoretic mobility similar to that of the CARP/DNA complex, and CARP binding to ClAde-containing oligomers was readily competed out by UV-irradiated DNA. Moreover, CARP activity was present in extracts prepared from UV-sensitive xeroderma pigmentosum group A cells but not in a subset of cells from group E, suggesting that CARP was similar to a previously described repair associated factor, xeroderma pigmentosum-E binding factor. Our findings support a possible repair process for ClAde residues incorporated into cellular DNA.

Induction of an apoptotic program in cell-free extracts by 2-chloro-2′-deoxyadenosine 5′-triphosphate and cytochrome c

“…The nucleotide inhibits ribonucleoside diphosphate reductase (25), DNA polymerases ␣ and ␤ (26), DNA ligase, and is incorporated into DNA (27). Together, these actions lead to the progressive accumulation of DNA single-strand breaks (20).…”

Analysis of 2-Chloro-2′-deoxyadenosine Incorporation into Cellular DNA by Quantitative Polymerase Chain Reaction

“…These interactions are thought to increase the fidelity of nucleotide incorporation at the polymerase active site (23), or to disrupt the water structure within the minor groove of B-DNA and create a larger minor groove width at the polymerase active site (24). The similarities between pol ␤ and Taq polymerase are of interest in light of our previous study in which we found that pol ␤ also could not bypass a template CldAMP residue under our standard in vitro DNA synthetic conditions (10). Thus, the presence of the chlorine group on Ade projecting out into the minor groove most likely prevents or modifies the above protein side chain-to-polymerase contact sites.…”

2-Chloro-2′-deoxyadenosine monophosphate residues in DNA enhance susceptibility to 3′ → 5′ exonucleases

“…Because CIdATP is not an absolute chain terminator for polymerase a, it is likely that its incorporation into cellular DNA will have significant effects on additional rounds ofDNA synthesis and enzymic processes. Indeed, we recently demonstrated that template ClAde residues were also in vitro blocks to DNA synthesis by human polymerases a and , (Hentosh and Grippo, 1994). These findings together with the above results may explain, in part, the reduced cellular DNA synthesis and may contribute to the cytotoxic action of CldAdo.…”