New Haven, CT, United States
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Rozacky J.,University of Texas at Austin | Nemec A.A.,Florida State University | Sweasy J.B.,The Yale Comprehensive Cancer Center | Kidane D.,University of Texas at Austin
Oncotarget | Year: 2015

DNA polymerase beta (Pol β) is a key enzyme for the protection against oxidative DNA lesions via its role in base excision repair (BER). Approximately 1/3 of tumors studied to date express Pol β variant proteins, and several tumors overexpress Pol β. Pol β possesses DNA polymerase and dRP lyase activities, both of which are known to be important for efficient BER. The dRP lyase activity resides within the 8kDa amino terminal domain of Pol β, is responsible for removal of the 5' phosphate group (5'-dRP). The DNA polymerase subsequently fills the gaps. Previously, we demonstrated that the human gastric cancer-associated variant of Pol β (Leu22Pro (L22P)) lacks dRP lyase function in vitro. Here, we report that L22P-expressing cells harbor significantly increased replication associated DNA double strand breaks (DSBs) and defective maintenance of the nascent DNA strand (NDS) during replication stress. Moreover, L22P-expressing cells are sensitive to PARP1 inhibitors, which suggests trapped PARP1 binds to the 5'-dRP group and blocks replications forks, resulting in fork collapse and DSBs. Our data suggest that the normal function of the dRP lyase is critical to maintain replication fork integrity and prevent replication fork collapse to DSBs and cellular transformation.


PubMed | University of Texas at Austin, The Yale Comprehensive Cancer Center and Florida State University
Type: Journal Article | Journal: Oncotarget | Year: 2015

DNA polymerase beta (Pol ) is a key enzyme for the protection against oxidative DNA lesions via its role in base excision repair (BER). Approximately 1/3 of tumors studied to date express Pol variant proteins, and several tumors overexpress Pol . Pol possesses DNA polymerase and dRP lyase activities, both of which are known to be important for efficient BER. The dRP lyase activity resides within the 8kDa amino terminal domain of Pol , is responsible for removal of the 5 phosphate group (5-dRP). The DNA polymerase subsequently fills the gaps. Previously, we demonstrated that the human gastric cancer-associated variant of Pol (Leu22Pro (L22P)) lacks dRP lyase function in vitro. Here, we report that L22P-expressing cells harbor significantly increased replication associated DNA double strand breaks (DSBs) and defective maintenance of the nascent DNA strand (NDS) during replication stress. Moreover, L22P-expressing cells are sensitive to PARP1 inhibitors, which suggests trapped PARP1 binds to the 5-dRP group and blocks replications forks, resulting in fork collapse and DSBs. Our data suggest that the normal function of the dRP lyase is critical to maintain replication fork integrity and prevent replication fork collapse to DSBs and cellular transformation.


PubMed | University of Texas at Austin and The Yale Comprehensive Cancer Center
Type: | Journal: Oncogenesis | Year: 2014

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.


Kidane D.,The Yale Comprehensive Cancer Center | Dalal S.,The Yale Comprehensive Cancer Center | Keh A.,The Yale Comprehensive Cancer Center | Liu Y.,The Yale Comprehensive Cancer Center | And 2 more authors.
DNA Repair | Year: 2011

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. © 2011 Elsevier B.V.


PubMed | The Yale Comprehensive Cancer Center
Type: Journal Article | Journal: The EMBO journal | Year: 2010

We have shown earlier that DNA polymerase beta (Pol beta) localizes to the synaptonemal complex (SC) during Prophase I of meiosis in mice. Pol beta 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 beta in recombination and/or synapsis, we used conditional gene targeting to delete the PolB gene from germ cells. We find that Pol beta-deficient spermatocytes are defective in meiotic chromosome synapsis and undergo apoptosis during Prophase I. We also find that SPO11-dependent gammaH2AX persists on meiotic chromatin, indicating that Pol beta is critical for the repair of SPO11-induced double-strand breaks (DSBs). Pol beta-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 beta-deficient spermatocyte nuclei. Localization of Pol beta to meiotic chromosomes requires the formation of SPO11-dependent DSBs. Taken together, these findings strongly indicate that Pol beta 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 beta-deficient spermatocytes are likely a direct consequence of these recombination defects.


PubMed | The Yale Comprehensive Cancer Center
Type: Journal Article | Journal: DNA repair | Year: 2011

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 ). We previously used conditional gene targeting to engineer mice with sperm deleted of the Pol B gene, which encodes Pol . 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 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 leads to increased frequencies and varieties of mutations. Our study also shows that Pol is critical for genome stability in the germline.

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