Immune Dysfunction in Mendelian Disorders of POLA1 Deficiency.

POLA1 encodes the catalytic unit of DNA polymerase α, which together with the Primase complex launches the DNA replication process. While complete deficiency of this essential gene is presumed to be lethal, at least two conditions due to partial POLA1 deficiency have been described. The first genetic syndrome to be mapped to POLA1 was X-linked reticulate pigmentary disorder (XLPDR, MIM #301220), a rare syndrome characterized by skin hyperpigmentation, sterile multiorgan inflammation, recurrent infections, and distinct facial features. XLPDR has been shown to be accompanied by profound activation of type I interferon signaling, but unlike other interferonopathies, it is not associated with autoantibodies or classical autoimmunity. Rather, it is accompanied by marked Natural Killer (NK) cell dysfunction, which may explain the recurrent infections seen in this syndrome. To date, all XLPDR cases are caused by the same recurrent intronic mutation, which results in gene missplicing. Several hypomorphic mutations in POLA1, distinct from the XLPDR intronic mutation, have been recently reported and these mutations associate with a separate condition, van Esch-O'Driscoll syndrome (VEODS, MIM #301030). This condition results in growth retardation, microcephaly, hypogonadism, and in some cases, overlapping immunological features to those seen in XLPDR. This review summarizes our current understanding of the clinical manifestations of POLA1 gene mutations with an emphasis on its immunological consequences, as well as recent advances in understanding of its pathophysiologic basis and potential therapeutic options.

Reducing DNA polymerase alpha in the absence of Drosophila ATR leads to P53-dependent apoptosis and developmental defects.

The ability to respond to DNA damage and incomplete replication ensures proper duplication and stability of the genome. Two checkpoint kinases, ATM and ATR, are required for DNA damage and replication checkpoint responses. In Drosophila, the ATR ortholog (MEI-41) is essential for preventing entry into mitosis in the presence of DNA damage. In the absence of MEI-41, heterozygosity for the E(mus304) mutation causes rough eyes. We found that E(mus304) is a mutation in DNApol-alpha180, which encodes the catalytic subunit of DNA polymerase alpha. We did not find any defects resulting from reducing Polalpha by itself. However, reducing Polalpha in the absence of MEI-41 resulted in elevated P53-dependent apoptosis, rough eyes, and increased genomic instability. Reducing Polalpha in mutants that lack downstream components of the DNA damage checkpoint (DmChk1 and DmChk2) results in the same defects. Furthermore, reducing levels of mitotic cyclins rescues both phenotypes. We suggest that reducing Polalpha slows replication, imposing an essential requirement for the MEI-41-dependent checkpoint for maintenance of genome stability, cell survival, and proper development. This work demonstrates a critical contribution of the checkpoint function of MEI-41 in responding to endogenous damage.

Characterization of Escherichia coli mutant strains deficient in AP DNA-repair synthesis.

Deficiency of apurinic/apyrimidinic (AP) DNA-repair enzymes in crude extracts of E. coli mutants was determined by following general and specific AP DNA-repair synthesis via nick translation in the presence of either all four dNTPs, or only one dNTP. We have shown that mutations either in DNA polymerase I or in AP endonucleases or in both, inhibit to different degrees the ability to repair AP DNA. The polA mutation totally abolishes the ability to perform both general and specific AP DNA repair, while the polAex mutation affects only general AP DNA repair. The xthA tight mutants, including the deletion mutant BW9101, can cope with small amounts of AP sites but hardly with high amounts of these lesions. In addition we have found that crude extracts of the xthA mutants degrade AP DNA by two modes: a nonspecific, and an AP-specific mode. These phenomena are common to all xth mutants and enabled us to discover this mutation. In contrast to the xth mutants so far isolated, BW2001 exhibits marked sensitivity to MMS and to X-ray irradiation. We found that this strain has a proficient DNA polymerase I but is absolutely deficient in AP endonucleases. We attribute its sensitivities to a secondary mutation at the structural gene of endonuclease IV.

Conditional lethality of the recA441 and recA730 mutants of Escherichia coli deficient in DNA polymerase I.

E. coli strains bearing the recA441 mutation and various mutations in the polA gene resulting in enzymatically well-defined deficiencies of DNA polymerase I have been constructed. It was found that the recA441 strains bearing either the polA1 or polA12 mutation causing deficiency of the polymerase activity of pol I are unable to grow at 42 degrees C on minimal medium supplemented with adenine, i.e., when the SOS response is continuously induced in strains bearing the recA441 mutation. Under these conditions the inhibition of DNA synthesis is followed in recA441 polA12 by DNA degradation and loss of cell viability. A similar lethal effect is observed with the recA730 polA12 mutant. The recA441 strain bearing the polA107 mutation resulting in the deficiency of the 5'-3' exonuclease activity of pol I shows normal growth under conditions of continuous SOS response. We postulate that constitutive expression of the SOS response leads to an altered requirement for the polymerase activity of pol I.

Mutagenicity of chloroalkene epoxides in bacterial systems.

6 alpha-chloroepoxides have been tested for in vitro activity in a variety of systems. The epoxides were cis- and trans-1-chloropropene oxide, cis- and trans-1,3-dichloropropene oxide, trichloroethylene oxide and tetrachloroethylene oxide. The epoxides were assayed for mutagenicity in the absence of metabolic activation in S. typhimurium TA1535 and E. coli WP2 uvrA and for preferential inhibition of growth of DNA-repair-deficient E. coli. All 6 epoxides possessed DNA-modifying activity as evidenced by their ability to preferentially inhibit DNA polymerase-deficient E. coli. All of the test chemicals except trichloroethylene oxide were mutagenic for S. typhimurium and all except trichloroethylene oxide and tetrachloroethylene oxide were mutagenic for E. coli Wp2 uvrA. Cis- and trans-1,3-dichloropropene oxide were the most potent mutagens and DNA modifiers. For all cases, the cis isomers were more active than the corresponding trans isomers. alpha-Chloroepoxides are considered likely to be the active intermediates of the carcinogenic parent halo-olefins. These mutagenicity studies are considered relevant in assessing the carcinogenicity of the parent hydrocarbons.

Induction of prophage and mutagenic effects by alkyl alkylaminosulfonates, ethyl aminosulfonate and alkyl methanesulfonates.

Prophage induction and mutation by alkylaminosulfonates, ethyl aminosulfonate and alkyl methanesulfonates were examined comparatively. Prophage induction was carried out with a lysozyme lysis technique on the lysogenic strain Micrococcus lysodeikticus 53-40 (N5). The sulfonic ester derivatives show a slight lysogenic induction. At higher concentrations their toxicity seems to mask phage detection. Only methyl isopropylaminosulfonate and ethyl aminosulfonate exhibit no or negligible toxic effects, and with these compounds at higher concentrations a strong prophage induction is found. Alkyl sulfonate derivatives induce mutations in the tester strain of Salmonella typhimurium TA1535. Methyl methylaminosulfonate and ethyl N-methyl-N-2-chloroethyl aminosulfonate show a mutagenicity comparable to that of the well-known methyl methanesulfonate or ethyl methanesulfonate. With ethyl aminosulfonate, however, which does not show inactivation, no significant mutagenic effect was observed. DNA alterations were found in the polymerase-deficient strain E. coli P3478. The results of prophage induction and mutagenicity are compared and discussed.

Identification of Escherichia coli dnaE (polC) mutants with altered sensitivity to 2',3'-dideoxyadenosine.

Bacteria with reduced DNA polymerase I activity have increased sensitivity to killing by chain-terminating nucleotides (S. A. Rashbaum and N. R. Cozzarelli, Nature 264:679-680, 1976). We have used this observation as the basis of a genetic strategy to identify mutations in the dnaE (polC) gene of Escherichia coli that alter sensitivity to 2',3'-dideoxyadenosine (ddA). Two dnaE (polC) mutant strains with increased sensitivity to ddA and one strain with increased resistance were isolated and characterized. The mutant phenotypes are due to single amino acid substitutions in the alpha subunit, the protein product of the dnaE (polC) gene. Increased sensitivity to ddA is produced by the L329F and H417Y substitutions, and increased resistance is produced by the G365S substitution. The L329F and H417Y substitutions also reduce the accuracy of DNA replication (the mutator phenotype), while the G365S substitution increases accuracy (the antimutator phenotype). All of the amino acid substitutions are in conserved regions near essential aspartate residues. These results prove the effectiveness of the genetic strategy in identifying informative dnaE (polC) mutations that can be used to elucidate the molecular basis of nucleotide interactions in the alpha subunit of the DNA polymerase III holoenzyme.