Mohiuddin, PhD - University of Toronto / The Hospital for Sick Children - Postdoctoral Fellow

Mohiuddin

PhD

University of Toronto / The Hospital for Sick Children

Postdoctoral Fellow

Toronto, Ontario | Canada

Main Specialties: Biology, Biotechnology, Medical Genetics, Medical Toxicology, Neurology


Top Author

Mohiuddin, PhD - University of Toronto / The Hospital for Sick Children - Postdoctoral Fellow

Mohiuddin

PhD

Introduction

MOHIUDDIN, PhD
Genetics & Genomic Biology, The Hospital for Sick Children,
University of Toronto, Canada

Education:
• Ph.D (April 2011 - March 2015)
Department of Hygiene and Public Health, Faculty of Medicine, Osaka Medical College, Japan. (I have done research in the Department of Radiation Genetics, Kyoto University with Professor Shunichi Takeda during my entire Ph.D.).

• Master of Science in Biochemistry & Molecular Biology (2008)
University of Dhaka, Bangladesh.

• Bachelor of Science (Honors) in Biochemistry & Molecular Biology (2006)
University of Dhaka, Bangladesh.

Training / Research Experiences:
• Visiting Researcher (September 2015 – November 2015)
Professor Yves Pommier’s lab, Laboratory of Molecular Pharmacology, Center for Cancer Research, NCI, NIH, Bethesda, MD, USA.
Research topic: DNA topoisomerases and DNA repair.

• Visiting Scientist (December 2014 –February 2015)
Dr. Julian Sale’s lab, MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, UK.
Research topic: DNA molecular combing.

• Visiting Student (August 2013 – November 2013)
Professor Roland Kanaar’s lab, Department of Genetics, Erasmus Medical Center, Rotterdam, Netherlands.
Research topics: CRISPR, TALEN and Phosphoproteome analysis.

• Visiting Student (December 2011)
Professor Seishiro Hirano’s lab, Center for Environmental Risk Research, NIES, Tsukuba, Japan.
Research project: Effect of carbon nanotubes on DNA repair.

Scientific achievements: During my PhD tenure, I conducted my research under the supervision of Professor Shunichi Takeda (Kyoto university, Japan), a well renowned scientist in DNA damage repair area. In my PhD and postdoc (one and half year) tenure in his lab, I have manipulated several genes in Human and chicken cell lines using CRISPR / TALEN and analyzed the role of these genes in the damage response pathways. In my first project, I have generated MARCO-Transferrin chimera to express in the gene-disrupted mutants and analyzed the DNA damage response to nano-particles such as carbon nanotubes induced DNA damage. It was published in “Archives of Toxicology” journal (2014). In my second project, I have analyzed the role of several E3 ligases in the ubiquitination pathway using gene-disrupted mutants and published this story in “DNA Repair” journal (2016). In my third project, I have analyzed the role of SUMO E3 ligases (PIAS1 and PIAS4) in the promotion of template switch by the SUMOylation of PCNA. This study is published recently in “PNAS” journal. In another project, I have generated several mis-match repair gene-disrupted human TK6 clones using CRISPR/TALEN and analyzed their role in homologous recombination. This manuscript will be submitted to “Nucleic Acids Research” journal soon. I have also contributed as a second author in other two publications in “Nucleic Acids Research” journal and as a co-author in several publications in peer-reviewed journals such as Cancer Research, PLoS One etc.

During my postdoc in The Hospital for Sick Children, Toronto, Canada, I have extended my research to find out the role of several damage repair genes in the maintenance of replication fork progression following chain terminating nucleosides analogs. The revised manuscript is submitted in “Nucleic Acids Research” journal. In another project, I am analyzing the role of damage repair genes in several neurodegenerative disorders. I am also assessing the levels of ADNP somatic mosaicism in DNA extracted from teeth, hair root cells (similar developmental origin as neurons) and ADNP patient-derived iPSCs of autistic offspring. I have contributed as a co-author in another work related to Autism and Alzheimer’s disease, which is under review in “Molecular Psychiatry” journal.

As to my experience fostering research collaborations, I developed collaborative links with Professor Roland Kanaar, Erasmus MC, Netherlands, Dr. Julian Sale, MRC - LMB, Cambridge, UK and Professor Yves Pommier, NCI, NIH, Bethesda, USA as the Visiting Scholar. At this point in time I am seeking to apply my experience and training in cell and molecular biology to a new and exciting field.

Primary Affiliation: University of Toronto / The Hospital for Sick Children - Toronto, Ontario , Canada

Specialties:

Research Interests:


View Mohiuddin’s Resume / CV

Education

Nov 2015
NIH, USA
Visiting Scientist
Dec 2014
MRC Lab, Cambridge, UK
Visiting Scientist
Aug 2013
Erasmus MC, Rotterdam, The Netherlands
Visiting Scientist

Publications

10Publications

397Reads

132Profile Views

86PubMed Central Citations

CtIP-BRCA1 complex and MRE11 maintain replication forks in the presence of chain terminating nucleoside analogs.

Nucleic Acids Res 2019 Jan 18. Epub 2019 Jan 18.

Program of Genetics and Genome Biology, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada.

Chain-terminating nucleoside analogs (CTNAs), which cannot be extended by DNA polymerases, are widely used as antivirals or anti-cancer agents, and can induce cell death. Processing of blocked DNA ends, like camptothecin-induced trapped-topoisomerase I, can be mediated by TDP1, BRCA1, CtIP and MRE11. Here, we investigated whether the CtIP-BRCA1 complex and MRE11 also contribute to cellular tolerance to CTNAs, including 2',3'-dideoxycytidine (ddC), cytarabine (ara-C) and zidovudine (Azidothymidine, AZT). We show that BRCA1-/-, CtIPS332A/-/- and nuclease-dead MRE11D20A/- mutants display increased sensitivity to CTNAs, accumulate more DNA damage (chromosomal breaks, ?-H2AX and neutral comets) when treated with CTNAs and exhibit significant delays in replication fork progression during exposure to CTNAs. Moreover, BRCA1-/-, CtIPS332A/-/- and nuclease-dead MRE11D20A/- mutants failed to resume DNA replication in response to CTNAs, whereas control and CtIP+/-/- cells experienced extensive recovery of DNA replication. In summary, we provide clear evidence that MRE11 and the collaborative action of BRCA1 and CtIP play a critical role in the nuclease-dependent removal of incorporated ddC from replicating genomic DNA. We propose that BRCA1-CTIP and MRE11 prepare nascent DNA ends, blocked from synthesis by CTNAs, for further repair.

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https://academic.oup.com/nar/advance-article/doi/10.1093/nar
Publisher Site
http://dx.doi.org/10.1093/nar/gkz009DOI Listing
January 2019
1 Read
11.561 Impact Factor

SUMOylation of PCNA by PIAS1 and PIAS4 promotes template switch in the chicken and human B cell lines.

Proc Natl Acad Sci U S A 2018 12 28;115(50):12793-12798. Epub 2018 Nov 28.

Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, 606-8501 Kyoto, Japan

DNA damage tolerance (DDT) releases replication blockage caused by damaged nucleotides on template strands employing two alternative pathways, error-prone translesion DNA synthesis (TLS) and error-free template switch (TS). Lys164 of proliferating cell nuclear antigen (PCNA) is SUMOylated during the physiological cell cycle. To explore the role for SUMOylation of PCNA in DDT, we characterized chicken DT40 and human TK6 B cells deficient in the PIAS1 and PIAS4 small ubiquitin-like modifier (SUMO) E3 ligases. DT40 cells have a unique advantage in the phenotypic analysis of DDT as they continuously diversify their immunoglobulin (Ig) variable genes by TLS and TS [Ig gene conversion (GC)], both relieving replication blocks at abasic sites without accompanying by DNA breakage. Remarkably, cells displayed a multifold decrease in SUMOylation of PCNA at Lys164 and over a 90% decrease in the rate of TS. Likewise, TK6 cells showed a shift of DDT from TS to TLS at a chemosynthetic UV lesion inserted into the genomic DNA. The mutation caused a ?90% decrease in the rate of Ig GC and no additional impact on cells. This epistatic relationship between the and the mutations suggests that PIAS1 and PIAS4 promote TS mainly through SUMOylation of PCNA at Lys164. This idea is further supported by the data that overexpression of a PCNA-SUMO1 chimeric protein restores defects in TS in cells. In conclusion, SUMOylation of PCNA at Lys164 promoted by PIAS1 and PIAS4 ensures the error-free release of replication blockage during physiological DNA replication in metazoan cells.

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http://dx.doi.org/10.1073/pnas.1716349115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6294928PMC
December 2018
233 Reads
2 Citations
9.809 Impact Factor

A High-Throughput Screen Identifies 2,9-Diazaspiro[5.5]Undecanes as Inducers of the Endoplasmic Reticulum Stress Response with Cytotoxic Activity in 3D Glioma Cell Models.

PLoS One 2016 29;11(8):e0161486. Epub 2016 Aug 29.

National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, 20850, United States of America.

The endoplasmic reticulum (ER) is involved in Ca2+ signaling and protein folding. ER Ca2+ depletion and accumulation of unfolded proteins activate the molecular chaperone GRP78 (glucose-regulated protein 78) which in turn triggers the ER stress response (ERSR) pathway aimed to restore ER homeostasis. Failure to adapt to stress, however, results in apoptosis. We and others have shown that malignant cells are more susceptible to ERSR-induced apoptosis than their normal counterparts, implicating the ERSR as a potential target for cancer therapeutics. Predicated on these findings, we developed an assay that uses a GRP78 biosensor to identify small molecule activators of ERSR in glioma cells. We performed a quantitative high-throughput screen (qHTS) against a collection of ~425,000 compounds and a comprehensive panel of orthogonal secondary assays was formulated for stringent compound validation. We identified novel activators of ERSR, including a compound with a 2,9-diazaspiro[5.5]undecane core, which depletes intracellular Ca2+ stores and induces apoptosis-mediated cell death in several cancer cell lines, including patient-derived and 3D cultures of glioma cells. This study demonstrates that our screening platform enables the identification and profiling of ERSR inducers with cytotoxic activity and advocates for characterization of these compound in in vivo models.

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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0161486PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5003374PMC
August 2017
37 Reads
3 Citations
3.234 Impact Factor

In vivo evidence for translesion synthesis by the replicative DNA polymerase δ.

Nucleic Acids Res 2016 09 16;44(15):7242-50. Epub 2016 May 16.

Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshidakonoe, Sakyo-ku, Kyoto 606-8501, Japan

The intolerance of DNA polymerase ? (Pol?) to incorrect base pairing contributes to its extremely high accuracy during replication, but is believed to inhibit translesion synthesis (TLS). However, chicken DT40 cells lacking the POLD3 subunit of Pol? are deficient in TLS. Previous genetic and biochemical analysis showed that POLD3 may promote lesion bypass by Pol? itself independently of the translesion polymerase Pol? of which POLD3 is also a subunit. To test this hypothesis, we have inactivated Pol? proofreading in pold3 cells. This significantly restored TLS in pold3 mutants, enhancing dA incorporation opposite abasic sites. Purified proofreading-deficient human Pol? holoenzyme performs TLS of abasic sites in vitro much more efficiently than the wild type enzyme, with over 90% of TLS events resulting in dA incorporation. Furthermore, proofreading deficiency enhances the capability of Pol? to continue DNA synthesis over UV lesions both in vivo and in vitro These data support Pol? contributing to TLS in vivo and suggest that the mutagenesis resulting from loss of Pol? proofreading activity may in part be explained by enhanced lesion bypass.

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https://academic.oup.com/nar/article-lookup/doi/10.1093/nar/
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http://dx.doi.org/10.1093/nar/gkw439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5009730PMC
September 2016
17 Reads
18 Citations
11.561 Impact Factor

The role of HERC2 and RNF8 ubiquitin E3 ligases in the promotion of translesion DNA synthesis in the chicken DT40 cell line.

DNA Repair (Amst) 2016 Apr 2;40:67-76. Epub 2016 Mar 2.

Department of Radiation Genetics, Kyoto University Graduate School of Medicine, Yoshida Konoe, Sakyo-ku, Kyoto 606-8501, Japan. Electronic address:

The replicative DNA polymerases are generally blocked by template DNA damage. The resulting replication arrest can be released by one of two post-replication repair (PRR) pathways, translesion DNA synthesis (TLS) and template switching by homologous recombination (HR). The HERC2 ubiquitin ligase plays a role in homologous recombination by facilitating the assembly of the Ubc13 ubiquitin-conjugating enzyme with the RNF8 ubiquitin ligase. To explore the role of HERC2 and RNF8 in PRR, we examined immunoglobulin diversification in chicken DT40 cells deficient in HERC2 and RNF8. Unexpectedly, the HERC2(-/-) and RNF8(-/-) cells and HERC2(-/-)/RNF8(-/-) double mutant cells exhibit a significant reduction in the rate of immunoglobulin (Ig) hypermutation, compared to wild-type cells. Further, the HERC2(-/-) and RNF8(-/-) mutants exhibit defective maintenance of replication fork progression immediately after exposure to UV while retaining proficient post-replicative gap filling. These mutants are both proficient in mono-ubiquitination of PCNA. Taken together, these results suggest that HERC2 and RNF8 promote TLS past abasic sites and UV-lesions at or very close to stalled replication forks.

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http://dx.doi.org/10.1016/j.dnarep.2016.02.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5351851PMC
April 2016
47 Reads
7 Citations
4.461 Impact Factor

Smarcal1 promotes double-strand-break repair by nonhomologous end-joining.

Nucleic Acids Res 2015 Jul 18;43(13):6359-72. Epub 2015 Jun 18.

Department of Radiation Genetics, Kyoto University, Graduate School of Medicine, Yoshida Konoe, Sakyo-ku, Kyoto 606-8501, Japan

Smarcal1 is a SWI/SNF-family protein with an ATPase domain involved in DNA-annealing activities and a binding site for the RPA single-strand-DNA-binding protein. Although the role played by Smarcal1 in the maintenance of replication forks has been established, it remains unknown whether Smarcal1 contributes to genomic DNA maintenance outside of the S phase. We disrupted the SMARCAL1 gene in both the chicken DT40 and the human TK6 B cell lines. The resulting SMARCAL1(-/-) clones exhibited sensitivity to chemotherapeutic topoisomerase 2 inhibitors, just as nonhomologous end-joining (NHEJ) null-deficient cells do. SMARCAL1(-/-) cells also exhibited an increase in radiosensitivity in the G1 phase. Moreover, the loss of Smarcal1 in NHEJ null-deficient cells does not further increase their radiosensitivity. These results demonstrate that Smarcal1 is required for efficient NHEJ-mediated DSB repair. Both inactivation of the ATPase domain and deletion of the RPA-binding site cause the same phenotype as does null-mutation of Smarcal1, suggesting that Smarcal1 enhances NHEJ, presumably by interacting with RPA at unwound single-strand sequences and then facilitating annealing at DSB ends. SMARCAL1(-/-)cells showed a poor accumulation of Ku70/DNA-PKcs and XRCC4 at DNA-damage sites. We propose that Smarcal1 maintains the duplex status of DSBs to ensure proper recruitment of NHEJ factors to DSB sites.

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http://dx.doi.org/10.1093/nar/gkv621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4513880PMC
July 2015
25 Reads
22 Citations
11.561 Impact Factor

A novel genotoxicity assay of carbon nanotubes using functional macrophage receptor with collagenous structure (MARCO)-expressing chicken B lymphocytes.

Arch Toxicol 2014 Jan 21;88(1):145-60. Epub 2013 Aug 21.

Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Kyoto, 606-8501, Japan.

Although carbon nanotubes (CNTs) are promising nanomaterials, their potential carcinogenicity is a major concern. We previously established a genetic method of analyzing genotoxicity of chemical compounds, where we evaluated their cytotoxic effect on the DT40 lymphoid cell line comparing DNA-repair-deficient isogenic clones with parental wild-type cells. However, application of our DT40 system for the cytotoxic and genotoxic evaluation of nanomaterials seemed to be difficult, because DT40 cells only poorly internalized nanoparticles. To solve this problem, we have constructed a chimeric gene encoding a trans-membrane receptor consisting of the 5' region of the transferrin receptor (TR) gene (to facilitate internalization of nanoparticles) and the 3' region of the macrophage receptor with collagenous structure (MARCO) gene (which is a receptor for environmental particles). We expressed the resulting MARCO-TR chimeric receptor on DNA-repair-proficient wild-type cells and mutants deficient in base excision repair (FEN1 (-/-)) and translesion DNA synthesis (REV3 (-/-)). We demonstrated that the chimera mediates uptake of particles such as fluorescence-tagged polystyrene particles and multi-walled carbon nanotubes (MWCNTs), with very poor uptake of those particles by DT40 cells not expressing the chimera. MWCNTs were cytotoxic and this effect was greater in FEN1 (-/-)and REV3 (-/-) cells than in wild-type cells. Furthermore, MWCNTs induced greater oxidative damage (measured as 8-OH-dG formation) and a larger number of mitotic chromosomal aberrations in repair-deficient cells compared to repair-proficient cells. Taken together, our novel assay system using the chimeric receptor-expressing DT40 cells provides a sensitive method to screen for genotoxicity of CNTs and possibly other nanomaterials.

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http://dx.doi.org/10.1007/s00204-013-1084-7DOI Listing
January 2014
5 Reads
10 Citations
5.980 Impact Factor

Precise determination of trace rubidium in biological fluid using inductively coupled plasma atomic emission spectroscopy

Bulletin of the Osaka Medical College 59(2):69-74

Rubidium (Rb) is a trace element that is ingested by humans daily, and also used in various industrial fields. However, there are few reports on the health effects caused by overexposure to Rb. In this study, we optimized the conditions for measuring Rb levels in biological fluid using inductively coupled plasma atomic emission spectroscopy (ICP—AES). The specific wavelength of 780.023 nm was used for the mea— surement. The Rb standard solution (1,000 ppm) was diluted to prepare 0, 100, 200, 300, and 500 pg/L Rb solutions, in order to determine the optimum wavelength, detection limit (DL; 44.1 pg/L), and quantitation limit (CML; 46.4 pg/L). To confirm the determination precision, Rb standard solution was added to rat urine with 50-fold dilution and human urine with 20-fold dilution, and the recovery rate of Rb was measured. In rat urine matrix, the recovery was 103.2 — 104.6%, and the coefficient of variation (%CV) was 0.39 — 0.80%. In human urine matrix, the recovery was 95.8 — 101.8%, and %CV was 0.47 — 0.86%. Thus, we confirmed that this examination has good precision and accuracy. ICP—AES can be applied to the assessment of toxicology and bioavailability of Rb in animal experiment or the spot urine screening of exposure to Rb even at early and low levels.

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December 2013

2 Citations

3 Reads

Structure-specific endonucleases xpf and mus81 play overlapping but essential roles in DNA repair by homologous recombination.

Cancer Res 2013 Jul 10;73(14):4362-71. Epub 2013 Apr 10.

Department of Radiation Genetics, and Frontier Technology Center, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto, Japan.

DNA double-strand breaks (DSB) occur frequently during replication in sister chromatids and are dramatically increased when cells are exposed to chemotherapeutic agents including camptothecin. Such DSBs are efficiently repaired specifically by homologous recombination (HR) with the intact sister chromatid. HR, therefore, plays pivotal roles in cellular proliferation and cellular tolerance to camptothecin. Mammalian cells carry several structure-specific endonucleases, such as Xpf-Ercc1 and Mus81-Eme1, in which Xpf and Mus81 are the essential subunits for enzymatic activity. Here, we show the functional overlap between Xpf and Mus81 by conditionally inactivating Xpf in the chicken DT40 cell line, which has no Mus81 ortholog. Although mammalian cells deficient in either Xpf or Mus81 are viable, Xpf inactivation in DT40 cells was lethal, resulting in a marked increase in the number of spontaneous chromosome breaks. Similarly, inactivation of both Xpf and Mus81 in human HeLa cells and murine embryonic stem cells caused numerous spontaneous chromosome breaks. Furthermore, the phenotype of Xpf-deficient DT40 cells was reversed by ectopic expression of human Mus81-Eme1 or human Xpf-Ercc1 heterodimers. These observations indicate the functional overlap of Xpf-Ercc1 and Mus81-Eme1 in the maintenance of genomic DNA. Both Mus81-Eme1 and Xpf-Ercc1 contribute to the completion of HR, as evidenced by the data that the expression of Mus81-Eme1 or Xpf-Ercc1 diminished the number of camptothecin-induced chromosome breaks in Xpf-deficient DT40 cells, and to preventing early steps in HR by deleting XRCC3 suppressed the nonviability of Xpf-deficient DT40 cells. In summary, Xpf and Mus81 have a substantially overlapping function in completion of HR.

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http://dx.doi.org/10.1158/0008-5472.CAN-12-3154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3718858PMC
July 2013
15 Reads
19 Citations
9.329 Impact Factor

Top co-authors

Shunichi Takeda
Shunichi Takeda

Graduate School of Medicine

8
Hiroyuki Sasanuma
Hiroyuki Sasanuma

Graduate School of Medicine

3
Kouji Hirota
Kouji Hirota

Graduate School of Science and Engineering

3
Md Maminur Rahman
Md Maminur Rahman

Kyoto University

3
Takeo Narita
Takeo Narita

Kyoto University

3
Kana Nishihara
Kana Nishihara

Kyoto University

3
Kelli Wilson
Kelli Wilson

Division of Preclinical Innovation

2
Kazutoshi Mori
Kazutoshi Mori

Graduate School of Science

2
Tetsuya Okada
Tetsuya Okada

Graduate School of Biostudies

2