Dawit Kidane

The RuvAB Branch Migration Translocase and RecU Holliday Junction Resolvase Are Required for Double-Stranded DNA Break Repair in Bacillus subtilis

In models of Escherichia coli recombination and DNA repair, the RuvABC complex directs the branch migration and resolution of Holliday junction DNA. To probe the validity of the E. coli paradigm, we examined the impact of mutations in ΔruvAB and ΔrecU (a ruvC functional analog) on DNA repair. Under standard transformation conditions we failed to construct ΔruvAB ΔrecG, ΔrecU ΔruvAB, ΔrecU ΔrecG, or ΔrecU ΔrecJ strains. However, ΔruvAB could be combined with addAB (recBCD), recF, recH, ΔrecS, ΔrecQ, and ΔrecJ mutations. The ΔruvAB and ΔrecU mutations rendered cells extremely sensitive to DNA-damaging agents, although less sensitive than a ΔrecA strain. When damaged cells were analyzed, we found that RecU was recruited to defined double-stranded DNA breaks (DSBs) and colocalized with RecN. RecU localized to these centers at a later time point during DSB repair, and formation was dependent on RuvAB. In addition, expression of RecU in an E. coli ruvC mutant restored full resistance to UV light only when the ruvAB genes were present. The results demonstrate that, as with E. coli RuvABC, RuvAB targets RecU to recombination intermediates and that all three proteins are required for repair of DSBs arising from lesions in chromosomal DNA.

DNA polymerase β is critical for mouse meiotic synapsis

We have shown earlier that DNA polymerase β (Pol β) localizes to the synaptonemal complex (SC) during Prophase I of meiosis in mice. Pol β localizes to synapsed axes during zygonema and pachynema, and it associates with the ends of bivalents during late pachynema and diplonema. To test whether these localization patterns reflect a function for Pol β in recombination and/or synapsis, we used conditional gene targeting to delete the PolB gene from germ cells. We find that Pol β‐deficient spermatocytes are defective in meiotic chromosome synapsis and undergo apoptosis during Prophase I. We also find that SPO11‐dependent γH2AX persists on meiotic chromatin, indicating that Pol β is critical for the repair of SPO11‐induced double‐strand breaks (DSBs). Pol β‐deficient spermatocytes yielded reduced steady‐state levels of the SPO11‐oligonucleotide complexes that are formed when SPO11 is removed from the ends of DSBs, and cytological experiments revealed that chromosome‐associated foci of replication protein A (RPA), RAD51 and DMC1 are less abundant in Pol β‐deficient spermatocyte nuclei. Localization of Pol β to meiotic chromosomes requires the formation of SPO11‐dependent DSBs. Taken together, these findings strongly indicate that Pol β is required at a very early step in the processing of meiotic DSBs, at or before the removal of SPO11 from DSB ends and the generation of the 3′ single‐stranded tails necessary for subsequent strand exchange. The chromosome synapsis defects and Prophase I apoptosis of Pol β‐deficient spermatocytes are likely a direct consequence of these recombination defects.

Mutation of POLB Causes Lupus in Mice

A replication study of a previous genome-wide association study (GWAS) suggested that a SNP linked to the POLB gene is associated with systemic lupus erythematosus (SLE). This SNP is correlated with decreased expression of Pol β, a key enzyme in the base excision repair (BER) pathway. To determine whether decreased Pol β activity results in SLE, we constructed a mouse model of POLB that encodes an enzyme with slow DNA polymerase activity. We show that mice expressing this hypomorphic POLB allele develop an autoimmune pathology that strongly resembles SLE. Of note, the mutant mice have shorter immunoglobulin heavy-chain junctions and somatic hypermutation is dramatically increased. These results demonstrate that decreased Pol β activity during the generation of immune diversity leads to lupus-like disease in mice, and suggest that decreased expression of Pol β in humans is an underlying cause of SLE.

The cell pole: the site of cross talk between the DNA uptake and genetic recombination machinery

Natural transformation is a programmed mechanism characterized by binding of free double-stranded (ds) DNA from the environment to the cell pole in rod-shaped bacteria. In Bacillus subtilis some competence proteins, which process the dsDNA and translocate single-stranded (ss) DNA into the cytosol, recruit a set of recombination proteins mainly to one of the cell poles. A subset of single-stranded binding proteins, working as “guardians”, protects ssDNA from degradation and limit the RecA recombinase loading. Then, the “mediators” overcome the inhibitory role of guardians, and recruit RecA onto ssDNA. A RecA·ssDNA filament searches for homology on the chromosome and, in a process that is controlled by “modulators”, catalyzes strand invasion with the generation of a displacement loop (D-loop). A D-loop resolvase or “resolver” cleaves this intermediate, limited DNA replication restores missing information and a DNA ligase seals the DNA ends. However, if any step fails, the “rescuers” will repair the broken end to rescue chromosomal transformation. If the ssDNA does not share homology with resident DNA, but it contains information for autonomous replication, guardian and mediator proteins catalyze plasmid establishment after inhibition of RecA. DNA replication and ligation reconstitute the molecule (plasmid transformation). In this review, the interacting network that leads to a cross talk between proteins of the uptake and genetic recombination machinery will be placed into prospective.

Germ-line variant of human NTH1 DNA glycosylase induces genomic instability and cellular transformation

Base excision repair (BER) removes at least 20,000 DNA lesions per human cell per day and is critical for the maintenance of genomic stability. We hypothesize that aberrant BER, resulting from mutations in BER genes, can lead to genomic instability and cancer. The first step in BER is catalyzed by DNA N-glycosylases. One of these, nth endonuclease III-like (NTH1), removes oxidized pyrimidines from DNA, including thymine glycol. The rs3087468 single nucleotide polymorphism of the NTH1 gene is a G-to-T base substitution that results in the NTH1 D239Y variant protein that occurs in ∼6.2% of the global population and is found in Europeans, Asians, and sub-Saharan Africans. In this study, we functionally characterize the effect of the D239Y variant expressed in immortal but nontransformed human and mouse mammary epithelial cells. We demonstrate that expression of the D239Y variant in cells also expressing wild-type NTH1 leads to genomic instability and cellular transformation as assessed by anchorage-independent growth, focus formation, invasion, and chromosomal aberrations. We also show that cells expressing the D239Y variant are sensitive to ionizing radiation and hydrogen peroxide and accumulate double strand breaks after treatment with these agents. The DNA damage response is also activated in D239Y-expressing cells. In combination, our data suggest that individuals possessing the D239Y variant are at risk for genomic instability and cancer.

Accumulation of abasic sites induces genomic instability in normal human gastric epithelial cells during Helicobacter pylori infection.

Helicobacter pylori infection of the human stomach is associated with inflammation that leads to the release of reactive oxygen and nitrogen species (RONs), eliciting DNA damage in host cells. Unrepaired DNA damage leads to genomic instability that is associated with cancer. Base excision repair (BER) is critical to maintain genomic stability during RONs-induced DNA damage, but little is known about its role in processing DNA damage associated with H. pylori infection of normal gastric epithelial cells. Here, we show that upon H. pylori infection, abasic (AP) sites accumulate and lead to increased levels of double-stranded DNA breaks (DSBs). In contrast, downregulation of the OGG1 DNA glycosylase decreases the levels of both AP sites and DSBs during H. pylori infection. Processing of AP sites during different phases of the cell cycle leads to an elevation in the levels of DSBs. Therefore, the induction of oxidative DNA damage by H. pylori and subsequent processing by BER in normal gastric epithelial cells has the potential to lead to genomic instability that may have a role in the development of gastric cancer. Our results are consistent with the interpretation that precise coordination of BER processing of DNA damage is critical for the maintenance of genomic stability.

In Vivo and In Vitro Evidence for Placental DNA Damage in Preeclampsia

Preeclampsia (PE) is an idiopathic multisystem disease affecting 5–7% of pregnant women. Placental oxidative stress is a characteristic feature of PE and occurs when the production of reactive oxygen species (ROS) within the placenta overwhelms the intrinsic anti-oxidant defenses. We hypothesize that excessive oxidative DNA damage at the fetal-maternal interface coupled with a defective DNA damage/repair response is causally related to PE. Here we demonstrate that γH2AX (a sensitive marker of DNA damage) is expressed in the maternal decidua but not trophoblast of normal placentas, and that expression is significantly higher in PE placental tissues in vivo. Using primary in vitro cultures of maternal decidual stromal cells (DSCs) and fetal cytotrophoblast cells (CTs), we show an increase in γH2AX foci in DSCs cultured with vs without H2O2 (70.6% vs 11.6%; P<0.0001) or under hypoxia-reperfusion vs normoxia (20- vs 3-fold; P = 0.01); no foci were seen in CTs. We further demonstrate that Base Excision Repair (BER) intermediates are significantly increased in DSCs (not CTs) under these same conditions. Our data show that DNA damage is significantly more common in PE placentas, and that this DNA damage is localized to the maternal and not fetal side of the placenta. CTs may be selectively resistant to DNA damage in an effort to protect the fetus.

DNA Polymerase Beta is Critical for Genomic Stability of Sperm Cells

Maintaining genome integrity in germ cells is important, given that the germ cells produce the next generation of offspring. Base excision repair is a DNA repair pathway that is responsible for the repair of most endogenous DNA damage. A key enzyme that functions in this repair pathway is DNA polymerase beta (Pol β). We previously used conditional gene targeting to engineer mice with sperm deleted of the Pol B gene, which encodes Pol β. We characterized mutagenesis in the sperm of these mice and compared it to wild-type and mice heterozygous for the Pol B gene. We found that sperm obtained that were heterozygously or homozygously deleted of the Pol B gene exhibited increased mutation frequencies compared to wild-type sperm. We identified an increase in transition mutations in both heterozygously and homozygously deleted sperm, and the types of mutations induced suggest that a polymerase other than Pol β functions in its absence. Interestingly, most of the transversions we observed were induced only in heterozygous, compared with wild-type sperm. Our results suggest that haploinsufficiency of Pol β leads to increased frequencies and varieties of mutations. Our study also shows that Pol β is critical for genome stability in the germline.

Kinesin 5B (KIF5B) Is Required for Progression through Female Meiosis and Proper Chromosomal Segregation in Mitotic Cells

The fidelity of chromosomal segregation during cell division is important to maintain chromosomal stability in order to prevent cancer and birth defects. Although several spindle-associated molecular motors have been shown to be essential for cell division, only a few chromosome arm-associated motors have been described. Here, we investigated the role of Kinesin 5b (Kif5b) during female mouse meiotic cell development and mitotic cell division. RNA interference (RNAi)-mediated silencing of Kif5b in mouse oocytes induced significant delay in germinal vesicle breakdown (GVBD) and failure in extrusion of the first polar body (PBE). In mitotic cells, knockdown of Kif5b leads to centrosome amplification and a chromosomal segregation defect. These data suggest that KIF5B is critical in suppressing chromosomal instability at the early stages of female meiotic cell development and mitotic cell division.