Mutational Signature Analysis Reveals NTHL1 Deficiency to Cause a Multi-tumor Phenotype.

Biallelic germline mutations affecting NTHL1 predispose carriers to adenomatous polyposis and colorectal cancer, but the complete phenotype is unknown. We describe 29 individuals carrying biallelic germline NTHL1 mutations from 17 families, of which 26 developed one (n = 10) or multiple (n = 16) malignancies in 14 different tissues. An unexpected high breast cancer incidence was observed in female carriers (60%). Mutational signature analysis of 14 tumors from 7 organs revealed that NTHL1 deficiency underlies the main mutational process in all but one of the tumors (93%). These results reveal NTHL1 as a multi-tumor predisposition gene with a high lifetime risk for extracolonic cancers and a typical mutational signature observed across tumor types, which can assist in the recognition of this syndrome.

Insights into the role of endonuclease V in RNA metabolism in Trypanosoma brucei.

Inosine may arise in DNA as a result of oxidative deamination of adenine or misincorporation of deoxyinosine triphosphate during replication. On the other hand, the occurrence of inosine in RNA is considered a normal and essential modification induced by specific adenosine deaminases acting on mRNA and tRNA. In prokaryotes, endonuclease V (EndoV) can recognize and cleave inosine-containing DNA. In contrast, mammalian EndoVs preferentially cleave inosine-containing RNA, suggesting a role in RNA metabolism for the eukaryotic members of this protein family. We have performed a biochemical characterization of EndoV from the protozoan parasite Trypanosoma brucei. In vitro, TbEndoV efficiently processes single-stranded RNA oligonucleotides with inosine, including A to I-edited tRNA-like substrates but exhibits weak activity over DNA, except when a ribonucleotide is placed 3' to the inosine. Immunolocalization studies performed in procyclic forms indicate that TbEndoV is mainly cytosolic yet upon nutritional stress it redistributes and accumulates in stress granules colocalizing with the DEAD-box helicase TbDhh1. RNAi-mediated depletion of TbEndoV results in moderate growth defects in procyclic cells while the two EndoV alleles could be readily knocked out in bloodstream forms. Taken together, these observations suggest an important role of TbEndoV in RNA metabolism in procyclic forms of the parasite.

The effect of Msh2 knockdown on toxicity induced by tert-butyl-hydroperoxide, potassium bromate, and hydrogen peroxide in base excision repair proficient and deficient cells.

The DNA mismatch repair (MMR) and base excision repair (BER) systems are important determinants of cellular toxicity following exposure to agents that cause oxidative DNA damage. To examine the interactions between these different repair systems, we examined whether toxicity, induced by t-BOOH and KBrO3, differs in BER proficient (Mpg (+/+), Nth1 (+/+)) and deficient (Mpg (-/-), Nth1 (-/-)) mouse embryonic fibroblasts (MEFs) following Msh2 knockdown of between 79 and 88% using an shRNA expression vector. Msh2 knockdown in Nth1 (+/+) cells had no effect on t-BOOH and KBrO3 induced toxicity as assessed by an MTT assay; knockdown in Nth1 (-/-) cells resulted in increased resistance to t-BOOH and KBrO3, a result consistent with Nth1 removing oxidised pyrimidines. Msh2 knockdown in Mpg (+/+) cells had no effect on t-BOOH toxicity but increased resistance to KBrO3; in Mpg (-/-) cells, Msh2 knockdown increased cellular sensitivity to KBrO3 but increased resistance to t-BOOH, suggesting a role for Mpg in removing DNA damage induced by these agents. MSH2 dependent and independent pathways then determine cellular toxicity induced by oxidising agents. A complex interaction between MMR and BER repair systems, that is, exposure dependent, also exists to determine cellular toxicity.

The DNA glycosylases Ogg1 and Nth1 do not contribute to Ig class switching in activated mouse splenic B cells.

During activation of B cells to undergo class switching, B cell metabolism is increased, and levels of reactive oxygen species (ROS) are increased. ROS can oxidize DNA bases resulting in substrates for the DNA glycosylases Ogg1 and Nth1. Ogg1 and Nth1 excise oxidized bases, and nick the resulting abasic sites, forming single-strand DNA breaks (SSBs) as intermediates during the repair process. In this study, we asked whether splenic B cells from mice deficient in these two enzymes would show altered class switching and decreased DNA breaks in comparison with wild-type mice. As the c-myc gene frequently recombines with the IgH S region in B cells induced to undergo class switching, we also analyzed the effect of deletion of these two glycosylases on DSBs in the c-myc gene. We did not detect a reduction in S region or c-myc DSBs or in class switching in splenic B cells from Ogg1- or Nth1-deficient mice or from mice deficient in both enzymes.

Closely opposed apurinic/apyrimidinic sites are converted to double strand breaks in Escherichia coli even in the absence of exonuclease III, endonuclease IV, nucleotide excision repair and AP lyase cleavage.

Multiply damaged sites (MDSs) consist of two or more damages within 20 base pairs (bps) and are introduced into DNA by ionizing radiation. Using a plasmid assay, we previously demonstrated that repair in Escherichia coli generated a double strand break (DSB) from two closely opposed uracils when uracil DNA glycosylase initiated repair. To identify the enzymes that converted the resulting apurinic/apyrimidinic (AP) sites to DSBs, repair was examined in bacteria deficient in AP site cleavage. Since exonuclease III (xth) and endonuclease IV (nfo) mutant bacteria were able to introduce DSBs at the MDSs, we generated unique bacterial mutants deficient in UvrA, Xth and Nfo. However, the additional disruption of nucleotide excision repair (NER) did not prevent DSB formation. xth- nfo- nfi- bacteria also converted the MDSs to DSBs, ruling out endonuclease V as the candidate AP endonuclease. By using MDSs containing tetrahydrofuran (an AP site analog), it was determined that even in the absence of Xth, Nfo, NER and AP lyase cleavage, DSBs were formed from closely opposed AP sites. This finding implies that there is an unknown enzyme/repair pathway for MDSs, and multiple underlying repair systems in cells that can process closely opposed DNA damage into lethal lesions following exposure to ionizing radiation.

Repair of formamidopyrimidines in DNA involves different glycosylases: role of the OGG1, NTH1, and NEIL1 enzymes.

The oxidatively induced DNA lesions 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) and 4,6-diamino-5-formamidopyrimidine (FapyA) are formed abundantly in DNA of cultured cells or tissues exposed to ionizing radiation or to other free radical-generating systems. In vitro studies indicate that these lesions are miscoding, can block the progression of DNA polymerases, and are substrates for base excision repair. However, no study has yet addressed how these lesions are metabolized in cellular extracts. The synthesis of oligonucleotides containing FapyG and FapyA at defined positions was recently reported. These constructs allowed us to investigate the repair of Fapy lesions in nuclear and mitochondrial extracts from wild type and knock-out mice lacking the two major DNA glycosylases for repair of oxidative DNA damage, OGG1 and NTH1. The background level of FapyG/FapyA in DNA from these mice was also determined. Endogenous FapyG levels in liver DNA from wild type mice were significantly higher than 8-hydroxyguanine levels. FapyG and FapyA were efficiently repaired in nuclear and mitochondrial extracts from wild type animals but not in the glycosylase-deficient mice. Our results indicated that OGG1 and NTH1 are the major DNA glycosylases for the removal of FapyG and FapyA, respectively. Tissue-specific analysis suggested that other DNA glycosylases may contribute to FapyA repair when NTH1 is poorly expressed. We identified NEIL1 in liver mitochondria, which could account for the residual incision activity in the absence of OGG1 and NTH1. FapyG and FapyA levels were significantly elevated in DNA from the knock-out mice, underscoring the biological role of OGG1 and NTH1 in the repair of these lesions.

Nei deficient Escherichia coli are sensitive to chromate and accumulate the oxidized guanine lesion spiroiminodihydantoin.

Growth inhibition and oxidized guanine lesion formation were studied in a number of base excision repair (BER) deficient Escherichia coli (E. coli) following chromate exposure. The only BER deficient bacterial strain that demonstrated significant growth inhibition by chromate, in comparison to its matched wild-type cell line, was the Nei deficient (TK3D11). HPLC coupled with electrospray ionization mass spectrometry showed that the Nei deficient E. coli accumulated the further oxidized guanine lesion, spiroiminodihydantoin (Sp), in genomic DNA at levels that were approximately 20-fold greater than its wild-type counterpart. However, no accumulation of the putative intermediate of Sp, 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG), was observed in the Nei deficient strain. A MutM-/MutY- double deletion mutant that was deficient in BER enzymes for the recognition and repair of 8-oxodG demonstrated no sensitivity toward chromate nor was there an associated increase in Sp accumulation over that of its wild type. However, the MutM-/MutY- double deletion mutant did show approximately 20-fold accumulation of 8-oxodG upon chromate exposure over that of the wild type and the Nei deficient E. coli. These data demonstrate that the Nei BER enzyme is critical for the recognition and repair of the Sp lesion in bacterial cell lines and demonstrates the protective effect of a specific BER enzyme on DNA lesions formed by chromate. To our knowledge, these are the first studies to show the formation and biological significance of the Sp lesion in a cellular system. This study has significant mechanistic and toxicological implications for how chromate may serve as an initiator of carcinogenesis and suggests a role for specific repair enzymes that may ameliorate the carcinogenic potential of chromate.