DNA polymerase β uses its lyase domain in a processive search for DNA damage

Abstract

DNA polymerase (Pol) β maintains genome fidelity by catalyzing DNA synthesis and removal of a reactive DNA repair intermediate during base excision repair (BER). Situated within the middle of the BER pathway, Pol β must efficiently locate its substrates before damage is exacerbated. The mechanisms of damage search and location by Pol β are largely unknown, but are critical for understanding the fundamental features of the BER pathway. We developed a processive search assay to determine if Pol β has evolved a mechanism for efficient DNA damage location. These assays revealed that Pol β scans DNA using a processive hopping mechanism and has a mean search footprint of ∼24 bp at predicted physiological ionic strength. Lysines within the lyase domain are required for processive searching, revealing a novel function for the lyase domain of Pol β. Application of our processive search assay into nucleosome core particles revealed that Pol β is not processive in the context of a nucleosome, and its single-turnover activity is reduced ∼500-fold, as compared to free DNA. These data suggest that the repair footprint of Pol β mainly resides within accessible regions of the genome and that these regions can be scanned for damage by Pol β.

Processive searching ability varies among members of the gap-filling DNA polymerase X family

“…It is important to discuss the interpretation of low F p values, such as the ones observed with Pol . Using our processive assay, the lowest F p value observed was 0.05, determined at 150 mM ionic strength with the K⌬3A Pol ␤ mutant (19). Similar low F p values have been obtained at high ionic strengths (0.08) Figure 2.…”

“…5A and supplemental Table S2). We previously showed that mutation of three lysines in the 8-kDa lyase domain of Pol ␤ to alanine (K35A, K68A, and K72A; referred to as K⌬3A) significantly reduced processive searching with the P20 substrate (19). This mutant similarly disrupted the ability of Pol ␤ to perform intersegmental transfer (Fig.…”

“…Processive searching, as measured here, is dependent on DNA scanning and nucleotide incorporation once a gap is encountered (i.e. product formation) (19). It is possible that a Pol may scan to a second site but dissociate before catalyzing nucleotide insertion, which would lead to a decrease in F p .…”

“…The F p value is the probability of an enzyme to perform a productive search, i.e. the ability to locate and catalyze product formation at both substrate sites within a single DNA-binding event (5,19). Representative gels for processive assays with Pols ␤ and are shown in Fig.…”

“…The DNA Pol X family contains four enzymes in humans: Pols ␤, , , and terminal deoxynucleotidyltransferase (TdT) (18). DNA Pol ␤ is critical in the BER pathway and has been demonstrated to perform a processive search (19). DNA Pols and are involved in nonhomologous end joining (NHEJ) with Pol having a backup role in BER (20,21).…”