The role of polymerase eta in somatic hypermutation determined by analysis of mutations in a patient with xeroderma pigmentosum variant

To determine the possible role of polymerase eta (pol eta) in somatic hypermutation of B cells, a mutational analysis of 24 nonproductive rearrangements from a patient with xeroderma pigmentosum variant with a defect in pol eta was conducted. Although the mutational frequency of A and T bases decreased in WA (A/T, A) motifs, regardless of their RGYW (purine, G; pyrimidine, A/T) context, the overall mutational frequency of A or T bases was not affected. Moreover, the overall mutational frequency of the sequences examined was not decreased. There was an apparent increase in the number of insertions and deletions. The results are consistent with the conclusion that pol eta specifically targets WA motifs. However, its overall contribution to the somatic hypermutational process does not appear to be indispensable and in its absence other mechanisms maintain mutational activity.     

DNA polymerases eta and theta function in the same genetic pathway to generate mutations at A/T during somatic hypermutation of Ig genes

Somatic hypermutation of the Ig genes requires the activity of multiple DNA polymerases to ultimately introduce mutations at both A/T and C/G base pairs. Mice deficient for DNA polymerase eta (POLH) exhibited an approximately 80% reduction of the mutations at A/T, whereas absence of polymerase (POLQ) resulted in approximately 20% reduction of both A/T and C/G mutations. To investigate whether the residual A/T mutations observed in the absence of POLH are generated by POLQ and how these two polymerases might cooperate or compete with each other to generate A/T mutations, here we have established mice deficient for both POLH and POLQ. Polq(-/-)Polh(-/-) mice, however, did not show a further decrease of A/T mutations as compared with Polh(-/-) mice, suggesting that POLH and POLQ function in the same genetic pathway in the generation of these mutations. Frequent misincorporation of nucleotides, in particular opposite template T, is a known feature of POLH, but the efficiency of extension beyond the misincorporation differs significantly depending on the nature of the mispairing. Remarkably, we found that POLQ catalyzed extension more efficiently than POLH from all types of mispaired termini opposite A or T. Moreover, POLQ was able to extend mispaired termini generated by POLH albeit at a relatively low efficiency. These results reveal genetic and biochemical interactions between POLH and POLQ and suggest that POLQ might cooperate with POLH to generate some of the A/T mutations during the somatic hypermutation of Ig genes.     

Crystal structures of 3-isopropylmalate dehydrogenases with mutations at the C-terminus: crystallographic analyses of structure-stability relationships

Thermal stability of the Thermus thermophilus isopropylmalate dehydrogenase enzyme was substantially lost upon the deletion of three residues from the C-terminus. However, the stability was partly recovered by the addition of two, four and seven amino acid residues (called HD177, HD708 and HD711, respectively) to the C-terminal region of the truncated enzyme. Three structures of these mutant enzymes were determined by an X-ray diffraction method. All protein crystals belong to space group P2(1) and their structures were solved by a standard molecular replacement method where the original dimer structure of the A172L mutant was used as a search model. Thermal stability of these mutant enzymes is discussed based on the 3D structure with special attention to the width of the active-site groove and the minor groove, distortion of beta-sheet pillar structure and size of cavity in the domain-domain interface around the C-terminus. Our previous studies revealed that the thermal stability of isopropylmalate dehydrogenase increases when the active-site cleft is closed (the closed form). In the present study it is shown that the active-site cleft can be regulated by open-close movement of the minor groove located at the opposite side to the active-site groove on the same subunit, through a paperclip-like motion.     

Base substitution mutations induced in the cI gene of lambda phage by neocarzinostatin chromophore: correlation with depyrimidination hotspots at the sequence AGC.

Treatment of intact lambda phage with the nonprotein chromophore of neocarzinostatin resulted in efficient phage inactivation and generation of clear-plaque mutants. Both effects required a preincubation at low pH to allow diffusion of chromophore into the phage head. Chromophore activation was then effected by addition of a sulfhydryl cofactor, followed by a shift to neutral pH. Sequence analysis of mutations mapped to the DNA-binding region of the cI gene revealed that nearly all were single base substitutions. Significant numbers of all possible base changes were found, with A:T to G:C transitions being the most frequent events. Of 11 G:C to A:T transitions, 7 were found at C residues in the trinucleotide sequence AGC, which has previously been shown to be a hotspot for chromophore-induced depyrimidination. This result, as well as the SOS dependence of mutagenesis and the overall distribution of various types of base substitutions, is consistent with the hypothesis that apurinic/apyrimidinic sites are important mutagenic lesions.