In vitro characterization of repair synthesis initiated by T4 endonuclease V on a synthetic DNA substrate.
The size of the repair patch produced by E. coli DNA polymerase (Pol I) following the removal of a pyrimidine dimer from DNA in response to the nicking activity of T4 endonuclease (T4 endo V) was determined. A 48-bp DNA containing a pyrimidine dimer at a defined location was labelled in the damaged strand and incubated with T4 endo V and E. coli endonuclease IV. Subsequently, DNA synthesis by DNA Pol I was carried out in the presence of four dNTPs, ATP and DNA ligase. Analysis of the reaction products on a sequencing gel revealed a ladder of only 4-oligonucleotides, 1-4 nucleotides greater in length than the fragment generated by the combined nicking activities of T4 endo V and E. coli endonuclease IV. Thus we conclude that the in vitro repair patch size of T4 endo V is 4 nucleotides and that in some cases the repaired DNA is not ligated.
Sibghat-Ullah
Department of Biochemistry and Biophysics, University of North Carolina, School of Medicine, Chapel Hill 27599.
Sancar
A
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D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004256:DNA Polymerase I / Q000378:metabolism*; D004260:DNA Repair; D004261:DNA Replication; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D008961:Models, Structural; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D013379:Substrate Specificity; D013604:T-Phages / Q000201:enzymology*; D014764:Viral Proteins
D009838:Oligodeoxyribonucleotides; D014764:Viral Proteins; D004247:DNA; D004256:DNA Polymerase I; D004706:Endodeoxyribonucleases; C470698:endonuclease V, phage T4; D043224:Deoxyribonuclease (Pyrimidine Dimer)