The fate of unrepaired BER intermediates caused by a dominant negative to DNA polymerase β Free

1453

Ionizing irradiation (IR) causes various types of DNA damage. The most frequent are base damages and single strand breaks (SSB). Less frequent but the most toxic are double strand breaks (DSB). The numerous lesions are predominantly repaired by the base excision repair (BER) and SSBR pathway, in which DNA polymerase beta (polbeta) has been shown to play a key role. We previously showed that inhibition of these repair processes by expressing a dominant negative to DNA polymerase (polDN) resulted in radiosensitization, confirming beta’s critical role in repair of IR-induced DNA damage. Increased radiosensitization could be due the presence of more toxic DSB caused by polDN by interference with the BER/SSBR pathways.
 In this study we wanted to address two questions. Firstly, does expression of polDN result in formation of secondary DSB, as is known for unrepaired BER/SSBR intermediates, leading to aberrations and death. Secondly, if more additional DSB are found, is this caused by increased formation of DSBs or inhibition of DSB repair.
 We found that expression of polDN led to an increased induction of chromosomal aberrations. These aberrations did not result from changes in induction or repair of the majority of DSB, as determined by constant field gel electrophoresis. The increase in aberrations after IR was mainly due to an increase of G1-type aberrations, indicating replication-independent formation of DSB. The increase in DSB was not, however, reflected in a parallel increase in γH2AX foci, since only a small increase in yH2AX foci in polDN expressing cells was observed compared to their vector controls. This suggests that the majority of DSB caused by the polDN are not recognized by the DSB repair machinery and therefore do not result in yH2AX foci. We further found that the polDN does not sensitize cells to H2O2, an agent producing oxidative base damage and SSB. This indicates that polDN is a specific sensitizer for IR, and suggests involvement in repair of clustered damage, which occurs after IR only.
 To address the second question, we tested polDN’s involvement in DSB repair using cell lines deficient in either homologous recombination (HR) or non-homologous endjoining (NHEJ). Preliminary data show that the observed increase in radiosensitization was not due to direct effects on repair of DSB by either of these pathways. These data also indicate a possible role for HR as a backup for repairing secondary DSB caused by polDN expression, since HR deficient cells show increased radiosensitivity when expressing the polDN, and also increased RAD51 foci. Specific inhibitors will be used to further exclude involvement of the polDN in DSB repair.
 We conclude that the polDN radiosensitizes by causing an increase in residual DSB which probably result from conversion of unrepaired SSB to DSB during replication or during repair attempts of clustered damaged sites. Moreover, these secondary DSB caused by polDN are dependent on HR for their repair.