Impairment of Proliferating Cell Nuclear Antigen-dependent Apurinic/Apyrimidinic Site Repair on Linear DNA

Samuel H. Wilson,Robert W. Sobol,Siham Biade,Yoshihiro Matsumoto

doi:10.1074/jbc.273.2.898

Abstract

Repair of apurinic/apyrimidinic (AP) sites by mammalian cell extracts was compared using circular and linear DNA substrates. Extracts prepared from DNA polymerase beta (polbeta)-proficient mouse fibroblasts repaired AP sites on both circular and linear DNA. However, extracts from the isogenic polbeta-knockout cells repaired AP sites on circular DNA but not efficiently on linear DNA. The circularity-dependent repair by the polbeta-knockout cell extract was completely inhibited by anti-proliferating cell nuclear antigen (PCNA) antibody but fully restored by addition of purified PCNA. Pretreatment of the linear DNA with AP endonuclease did not improve repair, indicating that impairment of AP site repair on linear DNA by polbeta-knockout cell extracts is not due to inefficiency of damage incision but rather to deficiency at the subsequent steps. These results indicate that AP sites can be repaired on circular DNA by the PCNA-dependent pathway in addition to the polbeta-dependent pathway and that the PCNA-dependent repair mechanism is poorly functional on linear DNA in vitro.

Highlights

Merases in nuclei, ␣, ␤, ␦, ⑀, and ␨ [3]
We have demonstrated that AP sites can be repaired by the pol␤-dependent pathway and an alternative pol␤-independent pathway in extracts prepared from mouse fibroblast-derived cells
The alternative pathway uses PCNA as one of its essential factors. This PCNA-dependent pathway was functional on circular DNA but not on linear DNA in vitro, whereas the pol␤-dependent repair reaction proceeded effi

Summary

Introduction

Merases in nuclei, ␣, ␤, ␦, ⑀, and ␨ [3]. Among them, pol␤ has long been considered to be involved in base excision repair. PCNA plays an essential role in DNA replication [8, 9] and in nucleotide excision repair [10, 11], and it has been shown recently to be involved in mismatch repair [12]. Unstable loading of the PCNA onto linear DNA is due to the falling off of the PCNA clamp from linear DNA ends after sliding along it If this property of PCNA-DNA binding applies to base excision repair, linear DNA substrates may not support the PCNA-dependent pathway. To test this possibility, we compared the repair efficiencies on circular and linear DNA side by side using extracts prepared from wild-type and pol␤-knockout mouse cells. We show here that DNA substrates that do not have free ends are preferable to detect PCNA-mediated in vitro repair

Methods

Results

Conclusion