In vitro characterization of repair synthesis initiated by T4 endonuclease V on a synthetic DNA substrate. |
| |
Authors: | Sancar" target="_blank">A Sibghat-UllahSancar |
| |
Institution: | Department of Biochemistry and Biophysics, University of North Carolina, School of Medicine, Chapel Hill 27599. |
| |
Abstract: | 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. |
| |
Keywords: | |
|
|