Apex1 can cleave complex clustered DNA lesions in cells

Abstract

Current data indicate that clustered DNA damage generated by ionizing radiation contains 2-5 damages within 20 bps. The complexity of clustered damage is also believed to increase as the linear energy transfer of the radiation increases. Complex lesions are therefore biologically relevant especially with the use of carbon ion beam therapy to treat cancer. Since two closely opposed AP site analogs (furans) are converted to a double strand break (DSB) in cells, we hypothesized that breakage could be compromised by increasing the complexity of the cluster. We have examined the repair of clusters containing three and four lesions in mouse fibroblasts using a luciferase reporter plasmid. The addition of a third furan did reduce but not eliminate cleavage, while a tandem 8-oxo-7,8 dihydroguanine (8oxoG) immediately 5' to one furan in a two or three furan cluster decreased DSB formation by a small amount. In vitro studies using nuclear extracts demonstrated that the tandem 8oxoG was not removed under conditions where the furan was cleaved, but the presence of the 8oxoG reduced cleavage at the furan. Interestingly, a cluster of an 8oxoG opposite a furan did not form a DSB in cells. We have shown that Apex1 can cleave these complex clustered lesions in cells. This therefore indicates that Apex1 can generate complex DSBs from clustered lesions consisting of base damage and AP sites. Repair of these complex DSBs may be compromised by the nearby oxidative damage resulting in potentially lethal and biologically relevant damage.

Distinct Roles of Ape1 Protein, an Enzyme Involved in DNA Repair, in High or Low Linear Energy Transfer Ionizing Radiation-induced Cell Killing

“…Repair of base damage is initiated by glycosylase (mainly Ogg1)-induced apurinic/apyrimidinic (AP) sites that are then cleaved by AP endonuclease (Ape1). Ape1 is an essential gene in mammalian cells and plays a protective role in low LET-irradiated cells (18,19), although it can also generate DSBs by dual cutting of closely opposed AP sites (19,20). Therefore, processing of clustered damage sites by Ogg1 and Ape1 may easily produce small DNA fragments.…”

Specificity of the dRP/AP Lyase of Ku Promotes Nonhomologous End Joining (NHEJ) Fidelity at Damaged Ends

“…6B, step ii). This is similar to the apparent discouragement of simultaneous action of base excision repair on nearby, opposite strand damaged sites within a damage cluster, a mechanism that helps reduce generation of DSBs (aborted base excision repair) (11)(12)(13). Indeed, Ku has been reported to inhibit AP site-cleaving activity when the AP site is embedded in double-stranded DNA (30,31).…”

“…Abasic sites are associated with chromosome breaks generated by exposure to ionizing radiation (as well as other sources of reactive oxygen species) (5, 10) and aborted base excision repair (BER) (11)(12)(13)(14) and associated with intermediates in immunoglobulin isotype class switch recombination (15,16). Such lesions are a strong block to ligation when present near a DSB terminus and must be excised (7)(8)(9)12).…”

Multiscale physics of ion-induced radiation damage

“…Complex DNA damage, investigated by biologists in Ward (1988Ward ( , 1995, Malyarchuk (2009), and Sage and Harrison (2011), is defined as a multiple number of primary DNA lesions happening on a length of two DNA twists. Such a correlation of lesions is called a cluster with a size equal to the number of singly damaged sites.…”