Energetics and Specificity of Rat DNA Polymerase β Interactions with Template-primer and Gapped DNA Substrates

Abstract

Interactions between rat polymerase ␤ (pol ␤) and the template-primer, as well as gapped DNAs, were studied using the quantitative fluorescence titration technique. Stoichiometries of rat pol ␤ complexes with DNA substrates are much higher than stoichiometries predicted by the structures of co-crystals. The data can be understood in the context of the two single-stranded (ss)DNAbinding modes of the enzyme, the (pol ␤ Polymerase ␤ is one of a number of recognized DNA-directed polymerases of the eukaryotic nucleus (1-5). The enzyme has a very specialized function in mammalian cell repair machinery. The activities of pol ␤ 1 have been proposed to include the gap filling synthesis involved in mismatch repair (4, 6 -8), in the repair of monofunctional adducts, UV damaged DNA, and abasic lesions in DNA (9 -14). Thus, a processive "gap fillings" synthesis, observed in vitro, by rat pol ␤ on gapped DNAs, is consistent with the proposed in vivo activities (2, 6 -8). A characteristic feature of rat pol ␤ is a "simplified" repertoire of activities. The enzyme is lacking intrinsic accessory activities, such as 3Ј-or 5Ј-exonuclease, endonuclease, dNMP turnover, and pyrophosphorolysis (1-4, 6, 7). Such limited activities reflect the very specialized function of the polymerase in the DNA metabolism.)The crystal structure of the rat pol ␤ enzyme revealed a typical polymerase fold, a thumb, palm, and fingers, due to its resemblance to the human hand (15-17). However, what distinguishes the pol ␤ structure from other polymerases is the presence of a small 8-kDa domain that is connected with the tip of the fingers through a tether of 14 amino acids (15-17). Solution studies showed that the 8-kDa domain has significant affinity for the single-stranded (ss) DNA, indicating that the domain plays a key role in recognition of this nucleic acid conformation, i.e. it is the template-binding domain (15,18,19,21,22). The active site of the DNA synthesis and the dsDNA affinity were proposed to reside predominantly in the large 31-kDa catalytic domain (15,18,19,23).Recently, we examined interactions of rat and human polymerase ␤ with the ssDNA (20, 24). Our data showed that rat pol ␤ binds the ssDNA in two binding modes which differ in the number of occluded nucleotide residues, the (pol ␤) 16 and (pol ␤) 5 binding mode. The binding modes differ in affinities and abilities to induce conformational changes in the ssDNA (20). Thus, the intrinsic affinity of the enzyme in the (pol ␤) 16 binding mode is approximately an order of magnitude higher than the affinity in the (pol ␤) 5 binding mode. On the other hand, when bound in the (pol ␤) 5 binding mode, the enzyme induces much more profound structural changes in the ssDNA, suggesting strong base-base separation and DNA immobilization in the complex (20). The obtained results also indicate that in the (pol ␤) 16 binding mode, both the 8-and 31-kDa domain of the enzyme, are involved in interactions with the ssDNA. In the (pol ␤) 5 binding mode, the 8-kDa domain predominately is engaged in interact...

A DNA polymerase β mutant from colon cancer cells induces mutations

“…Thus, once LOH occurs in the tumor, the WT pol ␤ would no longer be present to compete with K289M during BER, and we suggest that gap filling by K289M would lead to mutations. Second, pol ␤ is known to form multimeric complexes on DNA (44), and it has been proposed that this form of binding may stabilize pol ␤. Thus, in cells expressing equal amounts of WT and K289M, one would expect that a complex of K289M and WT would be bound to the gapped DNA, assuming that loading of these polymerases by apurinic͞apyrimidinic endonuclease is equally efficient, as suggested by the fact that they are both functional in the in vitro BER assay (Fig.…”

Dynamics of Gapped DNA Recognition by Human Polymerase β

“…Human Polymerase ␤-Human pol ␤ was purified as previously described (17)(18)(19)(20). The concentration of the protein was determined using the extinction coefficient, ⑀ 280 ϭ 2.1 ϫ 10 4 cm Ϫ1 M Ϫ1 , determined by the approach based on the Edelhoch method (23)(24)(25)(26)(27).…”

“…the substrates differ by three residues in the gap. This difference corresponds to the lowest estimate of the site-size of the (pol ␤) 5 binding mode (5 Ϯ 2) that the enzyme forms with the ssDNA (17)(18)(19)(20). The bases of the nucleotide residues in the ssDNA gap are all adenosines with the exception of the residue at the 5Ј end of the gap that is replaced by fluorescein.…”

“…Thus, the physiological substrates for the enzyme are gapped DNAs that have a stretch of ssDNA embedded between the primer and the dsDNA downstream from the primer. Thermodynamic studies of human pol ␤ binding to the gapped DNA substrates indicate that the ability of the 8-kDa domain DNA-binding site to interact with different nucleic acid conformations is crucial for anchoring the enzyme on the gap (19,20). In these complexes, the 8-kDa domain binds to the ss and/or dsDNA part of the DNA, downstream from the primer, depending on the length of the ssDNA gap.…”

“…Although the catalytic properties of the domains are established, the role of both domains in the recognition of the DNA substrates is just now emerging (17)(18)(19)(20). Quantitative equilibrium studies have shown that both human and rat pol ␤ bind the ssDNA in two binding modes (17,20).…”