Abstract
Xenopus oocytes and an oocyte nuclear extract efficiently repair the bulky DNA lesions cyclobutane pyrimidine dimers,(6-4) photoproducts, and N-acetoxy-2-aminofluorene (AAF) adducts by an excision repair mechanism. Nearly all (>95%) of the input damaged DNA was repaired within 5 h in both injected cells and extracts with no significant incorporation of label into control undamaged DNA. Remarkably, more than 10(10) cyclobutane pyrimidine dimers or(6-4) photoproducts are repaired/nuclei. The extracts are free from nuclease activity, and repair is independent of exogenous light. Both the high efficiency and DNA polymerase requirements of this system appear to be different from extracts derived from human cells. We demonstrated a requirement for DNA polymerases alpha and beta in repair of both photoproducts and AAF by inhibiting repair with several independent antibodies specific to either DNA polymerases alpha or beta and then restoring repair by adding the appropriate purified polymerase. Repair is inhibited by aphidicolin at concentrations specific for blocking DNA polymerase alpha and dideoxynucleotide triphosphates at concentrations specific for inhibiting DNA polymerase beta.
Highlights
DNA Polymerases ␣ and  Are Required for DNA Repair in an Efficient Nuclear Extract from Xenopus Oocytes*
Proteins involved in eukaryotic nucleotide excision repair (NER) have recently been identified [4], and the overall reaction has been reconstituted with purified proteins [7]
We demonstrate that a nuclear extract derived from Xenopus oocytes repairs Cyclobutane pyrimidine dimers (CPDs), the [] photoproduct, and the AAF lesion at an equivalent efficiency to that in injected cells, without significant incorporation of label into undamaged control DNA
Summary
Vol 271, No 23, Issue of June 7, pp. 13816 –13820, 1996 Printed in U.S.A. DNA Polymerases ␣ and  Are Required for DNA Repair in an Efficient Nuclear Extract from Xenopus Oocytes*. Xenopus oocytes and an oocyte nuclear extract efficiently repair the bulky DNA lesions cyclobutane pyrimidine dimers, [] photoproducts, and N-acetoxy-2aminofluorene (AAF) adducts by an excision repair mechanism. We demonstrate that a nuclear extract derived from Xenopus oocytes repairs CPD, the [] photoproduct, and the AAF lesion at an equivalent efficiency to that in injected cells, without significant incorporation of label into undamaged control DNA. Both DNA polymerases  and ␣ are required for the excision repair pathway of both photoproducts and AAF
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