Polµ tumor variants decrease the efficiency and accuracy of NHEJ.

The non homologous end-joining (NHEJ) pathway of double-strand break (DSB) repair often requires DNA synthesis to fill the gaps generated upon alignment of the broken ends, a complex task performed in human cells by two specialized DNA polymerases, Polλ and Polµ. It is now well established that Polµ is the one adapted to repair DSBs with non-complementary ends, the most challenging scenario, although the structural basis and physiological implications of this adaptation are not fully understood. Here, we demonstrate that two human Polµ point mutations, G174S and R175H, previously identified in two different tumor samples and affecting two adjacent residues, limit the efficiency of accurate NHEJ by Polµ in vitro and in vivo. Moreover, we show that this limitation is the consequence of a decreased template dependency during NHEJ, which renders the error-rate of the mutants higher due to the ability of Polµ to randomly incorporate nucleotides at DSBs. These results highlight the relevance of the 8 kDa domain of Polµ for accurate and efficient NHEJ, but also its contribution to the error-prone behavior of Polµ at 2-nt gaps. This work provides the first demonstration that mutations affecting Polµ identified in tumors can alter the efficiency and fidelity of NHEJ.

First principles calculations of the pKa values and tautomers of isoguanine and xanthine.

The accurate replication of DNA requires the formation of complementary hydrogen bonds between a template base and the base moiety of an incoming deoxynucleotide-5'-triphosphate. Recent structural studies suggest that some DNA polymerases contribute additional constraints by interrogating the minor groove face of the incoming and template bases. Therefore, the hydrogen bond-donating or -accepting properties of the base pairing as well as minor groove faces of the bases could be important determinants of correct base selection. In this paper, we investigate two purines that could arise by endogenous damage of the normal DNA bases: isoguanine (which can be generated by the oxidation of adenine) and xanthine (which can be generated by the deamination of guanine). In both cases, the potential exists for the placement of a proton in the N3 position, converting the N3 position from a hydrogen bond acceptor to a donor. In this paper, we use first principles quantum mechanical methods (density functional theory using the B3LYP functional and the 6-31G++Gbasis set) to predict the ionization and tautomeric equilibria of both isoguanine and xanthine in the gas phase and aqueous solution. For isoguanine, we find that the N1H and N3H neutral tautomeric forms are about equally populated in aqueous solution, while the enol tauotomers are predominant in the gas phase. In contrast, we find that xanthine displays essentially no tautomeric shifts in aqueous solution but is nearly equally populated by both an anionic and a neutral form at physiological pH. To obtain these results, we carried out an extensive examination of the tautomeric and ionic configurations for both xanthine and isoguanine in solution and in the gas phase. The potential hydrogen-bonding characteristics of these damaged purines may be used to test predictions of the important components of base selection by different DNA polymerases during DNA replication.

The beta subunit of the Escherichia coli DNA polymerase III holoenzyme interacts functionally with the catalytic core in the absence of other subunits.

We have previously demonstrated that the addition of a stoichiometric excess of the beta subunit of Escherichia coli DNA polymerase III holoenzyme to DNA polymerase III or holoenzyme itself can lead to an ATP-independent increase in the processivity of these enzyme forms (Crute, J. J., LaDuca, R. J., Johanson, K. O., McHenry, C. S., and Bambara, R. A. (1983) J. Biol. Chem. 258, 11344-11349). Here, we show that the beta subunit can interact directly with the catalytic core of the holoenzyme, DNA polymerase III, generating a new form of the enzyme with enhanced catalytic and processive capabilities. The addition of saturating levels of the beta subunit to the core DNA polymerase III enzyme results in as much as a 7-fold stimulation of synthetic activity. Two populations of DNA products were generated by the DNA polymerase III X beta enzyme complex. Short products resulting from the addition of 5-10 nucleotides/primer fragment were generated by DNA polymerase III in the presence and absence of added beta subunit. A second population of much longer products was generated only in beta-supplemented DNA polymerase III reactions. The DNA polymerase III-beta reaction was inhibited by single-stranded DNA binding protein and was unaffected by ATP, distinguishing it from the holoenzyme-catalyzed reaction. Complex formation of the DNA polymerase III core enzyme with beta increased the residence time of the enzyme on synthetic DNA templates. Our results demonstrate that the beta stimulation of DNA polymerase III can be attributed to a more efficient and highly processive elongation capability of the DNA polymerase III X beta complex. They also prove that at least part of beta's normal contribution to the DNA polymerase III holoenzyme reaction takes place through interaction with DNA polymerase III core enzyme components to produce the essential complex necessary for efficient elongation in vivo.