| 1. |
- Akuwudike, Pamela, 1987-, et al.
(author)
-
Mechanistic insights from high resolution DNA damage analysis to understand mixed radiation exposure
- 2023
-
In: DNA Repair. - 1568-7864 .- 1568-7856. ; 130
-
Journal article (peer-reviewed)abstract
- Cells exposed to densely ionising high and scattered low linear energy transfer (LET) radiation (50 % dose of each) react more strongly than to the same dose of each separately. The relationship between DNA double strand break location inside the nucleus and chromatin structure was evaluated, using high-resolution transmission electron microscopy (TEM) in breast cancer MDA-MB-231 cells at 30 min post 5 Gy. Additionally, response to high and/or low LET radiation was assessed using single (1 ×1.5 Gy) versus fractionated dose delivery (5 ×0.3 Gy). By TEM analysis, the highest total number of γH2AX nanobeads were found in cells irradiated with alpha radiation just prior to gamma radiation (called mixed beam), followed by alpha, then gamma radiation. γH2AX foci induced by mixed beam radiation tended to be surrounded by open chromatin (lighter TEM regions), yet foci containing the highest number of beads, i.e. larger foci representing complex damage, remained in the heterochromatic areas. The γH2AX large focus area was also greater in mixed beam-treated cells when analysed by immunofluorescence. Fractionated mixed beams given daily induced the strongest reduction in cell viability and colony formation in MDA-MB-231 and osteosarcoma U2OS cells compared to the other radiation qualities, as well as versus acute exposure. This may partially be explained by recurring low LET oxidative DNA damage by every fraction together with a delay in recompaction of chromatin after high LET, demonstrated by low levels of heterochromatin marker H3K9me3 at 2 h after the last mixed beam fraction in MDA-MB-231. In conclusion, early differences in response to complex DNA damage may lead to a stronger cell kill induced by fractionated exposure, which suggest a therapeutic potential of combined high and low LET irradiation.
|
|
| 2. |
- Berg, Ingrid L., et al.
(author)
-
MutS alpha deficiency increases tolerance to DNA damage in yeast lacking postreplication repair
- 2020
-
In: DNA Repair. - : Elsevier BV. - 1568-7864 .- 1568-7856. ; 91-92
-
Journal article (peer-reviewed)abstract
- By combining mutations in DNA repair genes, important and unexpected interactions between different repair pathways can be discovered. In this study, we identified a novel link between mismatch repair (MMR) genes and postreplication repair (PRR) in Saccharomyces cerevisiae. Strains lacking Rad5 (HLTF in mammals), a protein important for restarting stalled replication forks in the error-free PRR pathway, were supersensitive to the DNA methylating agent methyl methanesulfonate (MMS). Deletion of the mismatch repair genes, MSH2 or MSH6, which together constitutes the MutS alpha complex, partially suppressed the MMS super-sensitivity of the rad5 Delta, strain. Deletion of MSH2 also suppressed the MMS sensitivity of mms2 Delta, which acts together with Rad5 in error-free PRR. However, inactivating the mismatch repair genes MSH3 and MLH1 did not suppress rad5 Delta, showing that the suppression was specific for disabling MutS alpha. The partial suppression did not require translesion DNA synthesis (REV1, REV3 or RAD30), base excision repair (MAGI) or homologous recombination (RAD51). Instead, the underlying mechanism was dependent on RAD52 while independent of established pathways involving RAD52, like single-strand annealing and break-induced replication. We propose a Rad5- and Rad51-independent template switch pathway, capable of compensating for the loss of the error-free template-switch subpathway of postreplication repair, triggered by the loss of MutS alpha.
|
|
| 3. |
- Dmowski, Michal, et al.
(author)
-
Impairment of the non-catalytic subunit Dpb2 of DNA Pol ɛ results in increased involvement of Pol δ on the leading strand
- 2023
-
In: DNA Repair. - : Elsevier. - 1568-7864 .- 1568-7856. ; 129
-
Journal article (peer-reviewed)abstract
- The generally accepted model assumes that leading strand synthesis is performed by Pol ε, while lagging-strand synthesis is catalyzed by Pol δ. Pol ε has been shown to target the leading strand by interacting with the CMG helicase [Cdc45 Mcm2–7 GINS(Psf1–3, Sld5)]. Proper functioning of the CMG-Pol ɛ, the helicase-polymerase complex is essential for its progression and the fidelity of DNA replication. Dpb2p, the essential non-catalytic subunit of Pol ε plays a key role in maintaining the correct architecture of the replisome by acting as a link between Pol ε and the CMG complex. Using a temperature-sensitive dpb2–100 mutant previously isolated in our laboratory, and a genetic system which takes advantage of a distinct mutational signature of the Pol δ-L612M variant which allows detection of the involvement of Pol δ in the replication of particular DNA strands we show that in yeast cells with an impaired Dpb2 subunit, the contribution of Pol δ to the replication of the leading strand is significantly increased.
|
|
| 4. |
- Dmowski, Michal, et al.
(author)
-
Increased contribution of DNA polymerase delta to the leading strand replication in yeast with an impaired CMG helicase complex
- 2022
-
In: DNA Repair. - : Elsevier. - 1568-7864 .- 1568-7856. ; 110
-
Journal article (peer-reviewed)abstract
- DNA replication is performed by replisome proteins, which are highly conserved from yeast to humans. The CMG [Cdc45-Mcm2–7-GINS(Psf1–3, Sld5)] helicase unwinds the double helix to separate the leading and lagging DNA strands, which are replicated by the specialized DNA polymerases epsilon (Pol ε) and delta (Pol δ), respectively. This division of labor was confirmed by both genetic analyses and in vitro studies. Exceptions from this rule were described mainly in cells with impaired catalytic polymerase ε subunit. The central role in the recruitment and establishment of Pol ε on the leading strand is played by the CMG complex assembled on DNA during replication initiation. In this work we analyzed the consequences of impaired functioning of the CMG complex for the division labor between DNA polymerases on the two replicating strands. We showed in vitro that the GINSPsf1–1 complex poorly bound the Psf3 subunit. In vivo, we observed increased rates of L612M Pol δ-specific mutations during replication of the leading DNA strand in psf1–1 cells. These findings indicated that defective functioning of GINS impaired leading strand replication by Pol ε and necessitated involvement of Pol δ in the synthesis on this strand with a possible impact on the distribution of mutations and genomic stability. These are the first results to imply that the division of labor between the two main replicases can be severely influenced by a defective nonpolymerase subunit of the replisome.
|
|
| 5. |
|
|
| 6. |
|
|
| 7. |
- Keane, Simon, et al.
(author)
-
DLG2 impairs dsDNA break repair and maintains genome integrity in neuroblastoma
- 2022
-
In: DNA Repair. - : Elsevier. - 1568-7864 .- 1568-7856. ; 112
-
Journal article (peer-reviewed)abstract
- BackgroundIn primary neuroblastoma, deletions on chromosome 11q are known to result in an increase in the total number of chromosomal breaks. The DNA double-strand break repair pathways mediated by NHEJ are often upregulated in cancer. DLG2, a candidate tumor suppressor gene on chromosome 11q, has previously been implicated in DNA repair.MethodsWe evaluated an association between gene expression and neuroblastoma patient outcome, risk categorization, and 11q status using publicly available microarray data from independent neuroblastoma patient datasets. Functional studies were conducted using comet assay and H2AX phosphorylation in neuroblastoma cell lines and in the fruit fly with UVC-induced DNA breaks.ResultsWe show that the NHEJ genes PARP1 and FEN1 are over expressed in neuroblastoma and restoration of DLG2 impairs their gene and protein expression. When exposed to UVC radiation, cells with DLG2 over expression show less DNA fragmentation and induce apoptosis in a p53 S46 dependent manner. We could also confirm that DLG2 over expression results in CHK1 phosphorylation consistent with previous reports of G2/M maintenance.ConclusionsTaken together, we show that DLG2 over expression increases p53 mediated apoptosis in response to etoposide and UVC mediated genotoxicity and reduced DNA replication machinery.
|
|
| 8. |
- Singh, Vandana, 1985, et al.
(author)
-
Shining light on single-strand lesions caused by the chemotherapy drug bleomycin
- 2021
-
In: DNA Repair. - : Elsevier BV. - 1568-7864 .- 1568-7856. ; 105
-
Journal article (peer-reviewed)abstract
- Quantification of the DNA damage induced by chemotherapy in patient cells may aid in personalization of the dose used. However, assays to evaluate individual patient response to chemotherapy are not available today. Here, we present an assay that quantifies single-stranded lesions caused by the chemotherapeutic drug Bleomycin (BLM) in peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals. We use base excision repair (BER) enzymes to process the DNA damage induced by BLM and then extend the processed sites with fluorescent nucleotides using a DNA polymerase. The fluorescent patches are quantified on single DNA molecules using fluorescence microscopy. Using the assay, we observe a significant variation in the in vitro induced BLM damage and its repair for different individuals. Treatment of the cells with the BER inhibitor CRT0044876 leads to a lower level of repair of BLM-induced damage, indicating the ability of the assay to detect a compromised DNA repair in patients. Overall, the data suggest that our assay could be used to sensitively detect the variation in BLM-induced DNA damage and repair in patients and can potentially be able to aid in personalizing patient doses.
|
|
| 9. |
- Faraz, Mahmood, et al.
(author)
-
Tracking Escherichia coli DNA polymerase V to the entire genome during the SOS response
- 2021
-
In: DNA Repair. - : Elsevier BV. - 1568-7864. ; 101
-
Journal article (peer-reviewed)abstract
- Ribonucleotides are frequently incorporated into DNA and can be used as a marker of DNA replication enzymology. To investigate on a genome-wide scale, how E. coli pol V accesses undamaged chromosomal DNA during the SOS response, we mapped the location of ribonucleotides incorporated by steric gate variants of pol V across the entire E. coli genome. To do so, we used strains that are deficient in ribonucleotide excision repair (Delta rnhB), deficient in pol IV DNA polymerase, constitutively express all SOS-regulated genes [lexA(Def)] and constitutively "activated" RecA* (recA730). The strains also harbor two steric gate variants of E. coli pol V (Y11A, or F10L), or a homolog of pol V, (pol VR391-Y13A). Ribonucleotides are frequently incorporated by the pol V-Y11A and pol VR391-Y13A variants, with a preference to the lagging strand. In contrast, the pol V-F10L variant incorporates less ribonucleotides and no strand preference is observed. Sharp transitions in strand specificity are observed at the replication origin (oriC), while a gradient is observed at the termination region. To activate RecA* in a recA+ strain, we treated the strains with ciprofloxacin and genome-wide mapped the location of the incorporated ribonucleotides. Again, the pol V-Y11A steric gate variant exhibited a lagging strand preference. Our data are consistent with a specific role for pol V in lagging strand DNA synthesis across the entire E. coli genome during the SOS response.
|
|
