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The nature of the DNA substrate influences pre-catalytic conformational changes of DNA polymerase ?.

ABSTRACT: DNA polymerase ? (Pol ?) is essential for maintaining genomic integrity. During short-patch base excision repair (BER), Pol ? incorporates a nucleotide into a single-gapped DNA substrate. Pol ? may also function in long-patch BER, where the DNA substrate consists of larger gap sizes or 5'-modified downstream DNA. We have recently shown that Pol ? fills small gaps in DNA during microhomology-mediated end-joining as part of a process that increases genomic diversity. Our previous results with single-nucleotide gapped DNA show that Pol ? undergoes two pre-catalytic conformational changes upon binding to the correct nucleotide substrate. Here we use FRET to investigate nucleotide incorporation of Pol ? with various DNA substrates. The results show that increasing the gap size influences the fingers closing step by increasing its reverse rate. However, the 5'-phosphate group has a more significant effect. The absence of the 5'-phosphate decreases the DNA binding affinity of Pol ? and results in a conformationally more open binary complex. Moreover, upon addition of the correct nucleotide in the absence of 5'-phosphate, a slow fingers closing step is observed. Interestingly, either increasing the gap size or removing the 5'-phosphate group results in loss of the noncovalent step. Together, these results suggest that the character of the DNA substrate impacts the nature and rates of pre-catalytic conformational changes of Pol ?. Our results also indicate that conformational changes are important for the fidelity of DNA synthesis by Pol ?.

SUBMITTER: Huang J

PROVIDER: S-EPMC6166726 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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The nature of the DNA substrate influences pre-catalytic conformational changes of DNA polymerase β.

Huang Ji J Alnajjar Khadijeh S KS Mahmoud Mariam M MM Eckenroth Brian B Doublié Sylvie S Sweasy Joann B JB

The Journal of biological chemistry 20180801 39

DNA polymerase β (Pol β) is essential for maintaining genomic integrity. During short-patch base excision repair (BER), Pol β incorporates a nucleotide into a single-gapped DNA substrate. Pol β may also function in long-patch BER, where the DNA substrate consists of larger gap sizes or 5'-modified downstream DNA. We have recently shown that Pol β fills small gaps in DNA during microhomology-mediated end-joining as part of a process that increases genomic diversity. Our previous results with sing ...[more]

PMID: 30068550

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Project description:DNA polymerase (pol) β is a multidomain enzyme with two enzymatic activities that plays a central role in the overlapping base excision repair and single-strand break repair pathways. The high frequency of pol β variants identified in tumor-derived tissues suggests a possible role in the progression of cancer, making the determination of the functional consequences of these variants of interest. Pol β containing a proline substitution for leucine 22 in the lyase domain (LD), identified in gastric tumors, has been reported to exhibit severe impairment of both lyase and polymerase activities. Nuclear magnetic resonance (NMR) spectroscopic evaluations of both pol β and the isolated LD containing the L22P mutation demonstrate destabilization sufficient to result in LD-selective unfolding with minimal structural perturbations to the polymerase domain. Unexpectedly, addition of single-stranded or hairpin DNA resulted in partial refolding of the mutated lyase domain, both in isolation and for the full-length enzyme. Further, formation of an abortive ternary complex using Ca(2+) and a complementary dNTP indicates that the fraction of pol β(L22P) containing the folded LD undergoes conformational activation similar to that of the wild-type enzyme. Kinetic characterization of the polymerase activity of L22P pol β indicates that the L22P mutation compromises DNA binding, but nearly wild-type catalytic rates can be observed at elevated substrate concentrations. The organic osmolyte trimethylamine N-oxide (TMAO) is similarly able to induce folding and kinetic activation of both polymerase and lyase activities of the mutant. Kinetic data indicate synergy between the TMAO cosolvent and substrate binding. NMR data indicate that the effect of the DNA results primarily from interaction with the folded LD(L22P), while the effect of the TMAO results primarily from destabilization of the unfolded LD(L22P). These studies illustrate that substrate-induced catalytic activation of pol β provides an optimal enzyme conformation even in the presence of a strongly destabilizing point mutation. Accordingly, it remains to be determined whether this mutation alters the threshold of cellular repair activity needed for routine genome maintenance or whether the "inactive" variant interferes with DNA repair.

Dataset Information

The nature of the DNA substrate influences pre-catalytic conformational changes of DNA polymerase ?.

Publications

The nature of the DNA substrate influences pre-catalytic conformational changes of DNA polymerase β.

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