Off-target effects dominate a large-scale RNAi screen for modulators of the TGF-β pathway and reveal microRNA regulation of TGFBR2.

Background: RNA interference (RNAi) screens have been used to identify novel components of signal-transduction pathways in a variety of organisms. We performed a small interfering (si)RNA screen for novel members of the transforming growth factor (TGF)-β pathway in a human keratinocyte cell line. The TGF-β pathway is integral to mammalian cell proliferation and survival, and aberrant TGF-β responses have been strongly implicated in cancer.

Human AP endonuclease (APE1) demonstrates endonucleolytic activity against AP sites in single-stranded DNA.

Human apurinic/apyrimidinic endonuclease (APE1) is an enzyme of DNA base excision repair (BER) which catalyzes endonucleolytic cleavage immediately 5' to abasic (AP) sites. APE1 has long been thought to act on AP sites only in double stranded (ds) DNA, in order to generate the appropriate site for insertion of the correct nucleotide of DNA repair synthesis effected by DNA polymerase beta. We now present evidence that APE1 also acts on AP sites in single-stranded (ss) DNA.

Substrate specificity of human endonuclease III (hNTH1). Effect of human APE1 on hNTH1 activity.

Base excision repair of oxidized pyrimidines in human DNA is initiated by the DNA N-glycosylase/apurinic/apyrimidinic (AP) lyase, human NTH1 (hNTH1), the homolog of Escherichia coli endonuclease III (Nth). In contrast to Nth, the DNA N-glycosylase activity of hNTH1 is 7-fold greater than its AP lyase activity when the DNA substrate contains a thymine glycol (Tg) opposite adenine (Tg:A) (Marenstein, D. R.

Targeted deletion of mNth1 reveals a novel DNA repair enzyme activity.

DNA N-glycosylase/AP (apurinic/apyrimidinic) lyase enzymes of the endonuclease III family (nth in Escherichia coli and Nth1 in mammalian organisms) initiate DNA base excision repair of oxidized ring saturated pyrimidine residues. We generated a null mouse (mNth1(-/-)) by gene targeting. After almost 2 years, such mice exhibited no overt abnormalities.