title: Effects of DNA3'pp5'G capping on 3' end repair reactions and of an embedded pyrophosphate-linked guanylate on ribonucleotide surveillance.
abstract: When DNA breakage results in a 3'-PO4 terminus, the end is considered 'dirty' because it cannot prime repair synthesis by DNA polymerases or sealing by classic DNA ligases. The noncanonical ligase RtcB can guanylylate the DNA 3'-PO4 to form a DNA3'pp5'GOH cap. Here we show that DNA capping precludes end joining by classic ATP-dependent and NAD(+)-dependent DNA ligases, prevents template-independent nucleotide addition by mammalian terminal transferase, blocks exonucleolytic proofreading by Escherichia coli DNA polymerase II and inhibits proofreading by E. coli DNA polymerase III, while permitting templated DNA synthesis from the cap guanosine 3'-OH primer by E. coli DNA polymerase II (B family) and E. coli DNA polymerase III (C family). Human DNA polymerase β (X family) extends the cap primer predominantly by a single templated addition step. Cap-primed synthesis by templated polymerases embeds a pyrophosphate-linked ribonucleotide in DNA. We find that the embedded ppG is refractory to surveillance and incision by RNase H2.
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Chauleau
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.
authors.forename: Ushati
authors.initials: U
authors.lastname: Das
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA s-shuman@ski.mskcc.org.
authors.forename: Stewart
authors.initials: S
authors.lastname: Shuman
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D000818:Animals; D002417:Cattle; D053960:DNA Breaks; D059766:DNA End-Joining Repair; D011088:DNA Ligases / Q000378:metabolism; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D004257:DNA Polymerase II / Q000378:metabolism; D004258:DNA Polymerase III / Q000378:metabolism; D019951:DNA Polymerase beta / Q000378:metabolism; D004260:DNA Repair; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D016914:Ribonuclease H / Q000378:metabolism; D012265:Ribonucleotides / Q000737:chemistry / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D012265:Ribonucleotides; D006153:Guanosine Diphosphate; D004254:DNA Nucleotidyltransferases; D004257:DNA Polymerase II; D004258:DNA Polymerase III; D019951:DNA Polymerase beta; C091139:ribonuclease HII; D016914:Ribonuclease H; D000604:Amino Acyl-tRNA Synthetases; C561935:RtcB protein, E coli; D011088:DNA Ligases