Conversion of phosphoglycolate to phosphate termini on 3' overhangs of DNA double strand breaks by the human tyrosyl-DNA phosphodiesterase hTdp1.
Mammalian cells contain potent activity for removal of 3'-phosphoglycolates from single-stranded oligomers and from 3' overhangs of DNA double strand breaks, but no specific enzyme has been implicated in such removal. Fractionated human whole-cell extracts contained an activity, which in the presence of EDTA, catalyzed removal of glycolate from phosphoglycolate at a single-stranded 3' terminus to leave a 3'-phosphate, reminiscent of the human tyrosyl-DNA phosphodiesterase hTdp1. Recombinant hTdp1, as well as Saccharomyces cerevisiae Tdp1, catalyzed similar removal of glycolate, although less efficiently than removal of tyrosine. Moreover, glycolate-removing activity could be immunodepleted from the fractionated extracts by antiserum to hTdp1. When a plasmid containing a double strand break with a 3'-phosphoglycolate on a 3-base 3' overhang was incubated in human cell extracts, phosphoglycolate processing proceeded rapidly for the first few minutes but then slowed dramatically, suggesting that the single-stranded overhangs gradually became sequestered and inaccessible to hTdp1. The results suggest a role for hTdp1 in repair of free radical-mediated DNA double strand breaks bearing terminally blocked 3' overhangs.
Inamdar
Kedar V
KV
Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, VA 23298, USA.
Pouliot
Jeffrey J
JJ
Zhou
Tong
T
Lees-Miller
Susan P
SP
Rasouli-Nia
Aghdass
A
Povirk
Lawrence F
LF
D001483:Base Sequence; D015153:Blotting, Western; D002850:Chromatography, Gel; D004247:DNA / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000494:pharmacology; D006016:Glycolates / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010710:Phosphates / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013997:Time Factors
D006016:Glycolates; D010710:Phosphates; D011994:Recombinant Proteins; D004247:DNA; D004492:Edetic Acid; D010727:Phosphoric Diester Hydrolases; C498508:TDP1 protein, human; C122147:tyrosyl-DNA phosphodiesterase; C013193:phosphoglycolate