Lysines in the lyase active site of DNA polymerase β destabilize nonspecific DNA binding, facilitating searching and DNA gap recognition

Abstract

<jats:p>DNA polymerase (pol) b catalyzes two reactions at DNA gaps generated during base excision repair; gap-filling DNA synthesis and lyase-dependent 5' end deoxyribose phosphate removal. The lyase domain of pol β has been proposed to function in DNA gap recognition and to facilitate DNA scanning during substrate search. However, the mechanisms and molecular interactions used by pol b for substrate search and recognition are not clear. To provide insight into this process, a comparison was made of the DNA binding affinities of wild-type pol b, pol l and pol μ, and several variants of pol b, for 1-nt gap containing and undamaged DNA. Surprisingly, this analysis revealed that mutation of three lysine residues in the lyase active site of pol b, 35, 68 and 72, to alanine (pol b K∆3A), increased the binding affinity for non-specific DNA ~11-fold as compared to wild-type. Wild-type pol μ, lacking homologous lysines, displayed similar non-specific DNA binding behavior as pol b K∆3A, in line with previous data demonstrating both enzymes were deficient in processive searching. In fluorescent microscopy experiments using mouse fibroblasts deficient in PARP-1, the ability of pol b K∆3A to localize to sites of laser-induced DNA damage was strongly decreased, as compared to wild-type pol b. These data suggest that the three lysines in the lyase active site destabilize pol b when bound to DNA non-specifically, promoting DNA scanning and providing binding specificity for gapped DNA.</jats:p>

Human Polβ Natural Polymorphic Variants G118V and R149I Affects Substate Binding and Catalysis

“…During this analysis, the formation of several complexes with different mobility in the gel was recorded. Of note, the formation of several complexes with different gel mobility in the EMSA has also been reported earlier [ 48 , 49 , 50 ]. It is possible that Polβ forms complexes with DNA in some fixed intermediate states between the well-known open and closed conformations, and these intermediate states possess different electrophoretic mobility.…”

T. cruzi DNA polymerase beta (Tcpolβ) is phosphorylated in vitro by CK1, CK2 and TcAUK1 leading to the potentiation of its DNA synthesis activity

“…Also, the phosphorylation might cause changes in the subcellular location of Tcpolβ, since it has been reported that in hydrogen peroxide treated epimastigote cells, Tcpolβ appears in new additional focus outside of the kDNA as compared to untreated cells [18]. It has been recently shown that mammalian DNA polymerase β can perform a processive search (DNA scanning) looking for DNA damage using its lyase domain [29,30]. This indicates that the dRP lyase domain of DNA polymerase β can non-specifically interact with DNA during the scanning process.…”

When DNA Polymerases Multitask: Functions Beyond Nucleotidyl Transfer

“…Recently, it was found that three lysines in the lyase active site of Pol β facilitate the search for the 5′-flap substrate on the dsDNA and destabilize non-specific DNA binding. Mutating these three lysine residues to alanines in the lyase active site of Pol β (Lys 35, Lys 68, and Lys 72), increased the binding affinity of Pol β for nonspecific DNA, indicating a role of the 5′dRP lyase in the specificity of lesion recognition (Howard et al, 2020). A tumor-associated variant in the N-terminal 8 kDa domain of Pol β retains polymerase activity but is dRP lyase deficient (Dalal et al, 2008).…”