The zinc fingers of human poly(ADP-ribose) polymerase are differentially required for the recognition of DNA breaks and nicks and the consequent enzyme activation. Other structures recognize intact DNA.
The recognition of double-stranded DNA breaks and single-stranded nicks by human poly(ADP-ribose) polymerase and the consequent enzymic activation were examined using derivatives of the enzyme expressed in Escherichia coli. The N-terminal 162 residues encompass two zinc fingers. Deletion or mutation of the first finger results in a loss of activation by DNA with either single-stranded or double-stranded damage. Destruction of the second finger reduces activation by double-stranded DNA breaks only slightly, but eliminates activation by single-stranded DNA nicks. These data suggest that activation by single-stranded DNA nicks requires two zinc fingers, but activation by double-stranded DNA breaks requires only the finger closer to the N terminus. Variant proteins that lack both zinc fingers are enzymically inactive but still exhibit weak DNA binding, which is independent of DNA damage. Thus, other regions are also capable of binding intact DNA, but the recognition of a strand nick or break which occasions the synthesis of poly(ADP-ribose) specifically requires the zinc fingers.
Ikejima
M
M
Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
Noguchi
S
S
Yamashita
R
R
Ogura
T
T
Sugimura
T
T
Gill
D M
DM
Miwa
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003545:Cysteine / Q000502:physiology; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D004252:DNA Mutational Analysis; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D008969:Molecular Sequence Data; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases; D003545:Cysteine