Papers by Mahmud K . K . Shivji
A thermostable endonuclease III homolog from
Molecular and Cellular Biology, Mar 1, 1991
Murine F9 embryonal carcinoma (F9 EC) stem cells have an Ela-like transcription activity that is ... more Murine F9 embryonal carcinoma (F9 EC) stem cells have an Ela-like transcription activity that is downregulated as these cells differentiate to parietal endoderm. For the adenovirus E2A promoter, this activity requires at least two sequence-specific transcription factors, one that binds the cyclic AMP-responsive element (CRE) and the other, DRTF1, the DNA-binding activity of which is down-regulated as F9 EC cells differentiate. Here we report the characterization of several binding activities in F9 EC cell extracts, referred to as DRTF la, lb and lc, that recognize the DRTF1 cis-regulatory sequence (-70 to-50 region). These activities can be chromatographically separated but are not distinguishable by DNA sequence specificity. Activity la is a detergent-sensitive complex in which DNA binding is regulated by phosphorylation. In contrast, activities lb and lc are unaffected by these treatments but exist as multicomponent protein complexes even before DNA binding. Two sets of DNA-binding polypeptides, p50DR and p3ODR, affinity purified from F9 EC cell extracts produce complexes lb and lc. Both polypeptides appear to be present in the same DNA-bound protein complex and both directly contact DNA. These affinity-purified polypeptides activate transcription in vitro in a binding-site-dependent manner. These data indicate the in F9 EC stem cells, multicomponent differentiationregulated transcription factors contribute to the cellular Ela-like activity.
Protein expression and purification. BRC4, ∆1524-30 peptides (Figure S1A) and T1526A mutant pepti... more Protein expression and purification. BRC4, ∆1524-30 peptides (Figure S1A) and T1526A mutant peptide were cloned into pGEX-6-1 (Amersham Biosciences), and the GST-tagged BRC4, ∆1524-30 and T1526A fusions were expressed in BL21 (DE3) cells. For each protein, about 8 g of cell paste from 3 liters of culture were suspended in 40 ml of PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.3) supplemented with 1 mM PMSF. The cells were disrupted using a French press. The crude lysate was clarified by centrifugation in a Beckmann Ti 45 rotor 35,000 rpm, 60 min, and the cleared lysate was applied to a 5 ml GSTrap HP column (Amersham) equilibrated with PBS buffer. The GST-fusion protein was eluted with 10 mM reduced glutathione and 50 mM TrisHCl (pH 8.0). The protein sample was then dialyzed against a buffer containing: 20 mM TrisHCl (pH 7.5), 1mM EDTA, 1mM dithiothreitol (DTT), and 10 % glycerol, and applied into a 6 ml Resource Q column (Amersham). The protein was eluted wit...
A region of human BRCA2 containing multiple BRC repeats promotes RAD51-mediated
Molecular and Cellular Biology, 1991
Murine F9 embryonal carcinoma (F9 EC) stem cells have an E1a-like transcription activity that is ... more Murine F9 embryonal carcinoma (F9 EC) stem cells have an E1a-like transcription activity that is down-regulated as these cells differentiate to parietal endoderm. For the adenovirus E2A promoter, this activity requires at least two sequence-specific transcription factors, one that binds the cyclic AMP-responsive element (CRE) and the other, DRTF1, the DNA-binding activity of which is down-regulated as F9 EC cells differentiate. Here we report the characterization of several binding activities in F9 EC cell extracts, referred to as DRTF 1a, 1b and 1c, that recognize the DRTF1 cis-regulatory sequence (-70 to -50 region). These activities can be chromatographically separated but are not distinguishable by DNA sequence specificity. Activity 1a is a detergent-sensitive complex in which DNA binding is regulated by phosphorylation. In contrast, activities 1b and 1c are unaffected by these treatments but exist as multicomponent protein complexes even before DNA binding. Two sets of DNA-bind...
A region of human BRCA2 containing multiple BRC repeats promotes RAD51-mediated
Haematology and blood transfusion, 1989
Translocation t(9; 22) (q34; q11) occurs in 90% of patients with chronic myeloid leukaemia (CML) ... more Translocation t(9; 22) (q34; q11) occurs in 90% of patients with chronic myeloid leukaemia (CML) [13] and in 5% of children and 10%–20% of adults with acute lymphoblastic leukaemia (ALL) [16]. It is easily identified by the 22q−, or Philadelphia, chromosome. In CML all chromosome 22 breakpoints are located within the 5.8-kb breakpoint cluster region (bcr) in the 3′ part of the phl gene [6]. In ALL, however, only some breakpoints are in bcr. Others are more 5′ in the phl gene, as suggested by observations made at the RNA and protein level [3, 4, 9, 17] and at the DNA level by pulsed-field gel electrophoresis [14]. Both in CML and in ALL the breakpoint on chromosome 9 is within the abl oncogene upstream of the common exon [7].
Blood, 1988
The Philadelphia (Ph) translocation t(9;22)(q34;q11) occurs frequently in chronic myeloid leukemi... more The Philadelphia (Ph) translocation t(9;22)(q34;q11) occurs frequently in chronic myeloid leukemia (CML) but is less common in acute lymphoblastic leukemia (ALL) and rare in acute myeloid leukemia (AML). In most cases of CML and some cases of Ph+ ALL the protooncogene ABL from 9q34 is translocated to the breakpoint cluster region (bcr) of the BCR gene at 22q11 to form a chimeric gene encoding a novel 210-kd protein (P210 BCR-ABL) with enhanced tyrosine kinase activity. In other patients with Ph+ ALL and Ph+ AML, the breakpoint probably occurs in the first intron of the BCR gene; this results in a smaller chimeric gene which encodes a P190 BCR-ABL. We studied a patient with AML (FAB M6) arising de novo who had a “masked” Ph chromosome in association with extensive karyotypic changes. The leukemic cells initially showed rearrangement of the bcr, presence of a hybrid mRNA, and expression of the P210 BCR-ABL. These changes were absent in remission. These results support the concept that...
Blood, 1988
The chromosome 22 derivative, the Philadelphia (Ph) chromosome, results from a reciprocal translo... more The chromosome 22 derivative, the Philadelphia (Ph) chromosome, results from a reciprocal translocation t(9;22) (q34;q11) and is associated with chronic myeloid leukemia (CML). The translocation can be identified at the DNA level in Ph-positive CML by using a probe to the breakpoint cluster region (bcr). In addition, as a result of this translocation an abl-related 210-kd protein with protein tyrosine kinase (PTK) activity is produced. We analyzed 28 cases of Ph-negative CML for rearrangement of the chromosome 22 sequences and found that eight of the 28 show rearrangement of the bcr. When 12 of the Ph- negative cases were independently reviewed, five were indistinguishable from Ph-positive CML on the basis of morphology, peripheral blood film and clinical details. These five also showed bcr rearrangement. The other seven were reclassified as six atypical CML (aCML) and one chronic myelomonocytic leukemia (CMML). None of these seven showed bcr rearrangement. In addition 11 cases of b...
Oncogene, 1988
Approximately 5% of children and 10-20% of adults with acute lymphoblastic leukaemia (ALL) have a... more Approximately 5% of children and 10-20% of adults with acute lymphoblastic leukaemia (ALL) have a chromosome translocation t(9;22) which at the cytogenetic level appears identical to that in chronic myeloid leukaemia (CML). The t(9;22) translocation was first recognised in CML patients by its 22q- or Philadelphia (Ph) chromosome. While all Ph positive CML patients so far described have a chromosome 22 breakpoint within the breakpoint cluster region (bcr) located in the 3' part of the phl gene, only some Ph positive ALL patients have breakpoints in bcr. We have cloned the breakpoint of the 9q+ chromosome from the DNA of a Ph positive ALL patient in whom there is no breakpoint in the bcr. The non-chromosome 9 sequences of the breakpoint region are shown to be derived from chromosome 22. The breakpoint in chromosome 22 is shown to be the first intron of the phl gene about 66kb upstream of the bcr. Using probes from this intron, rearrangements were detected in the DNA of two out of ...
Nucleic Acids Research, 1989
Breakpoints on chromosome 22 in the translocation t(9;22) found in Philadelphia positive acute r ... more Breakpoints on chromosome 22 in the translocation t(9;22) found in Philadelphia positive acute r lymphoblastic leukaemia patients fall within two categories. In the first the breakpoint is localized within the breakpoint cluster region of the BCR gene, analogous to the chromosome 22 breakpoint in chronic myeloid leukaemia. The second category has a breakpoint 5' of this area, but still within the BCR gene. We have previously shown that these breakpoints occur within the first intron of the BCR gene and cloned the 9q + junction from such a patient. We have now determined the * sequences around the breakpoints on both translocation partners from this patient as well as the germline regions. The chromosome 9 ABL sequence around the breakpoint shows homology to the consensus t-Alu sequence whereas the chromosome 22 BCR sequence does not. At the junction there is a 6 bp duplication of the chromosome 22 sequence which is present both in the 9q + and in the 22q* translocation products. Possible mechanisms for the generation of the translocation are discussed.
ABSTRACT Thesis (Ph. D.)--Open University, 1996.
Journal of Biological Chemistry, 1994
I " A predominant form of the inherited syndrome xeroderma pigmentosum is genetic complementation... more I " A predominant form of the inherited syndrome xeroderma pigmentosum is genetic complementation group C (XP-C). XP-C cells are defective in DNA nucleotide excision repair in the bulk of the genome but can repair transcribed strands of active genes. An activity that can complement the repair deficiency of extracts from Xp-C cells has been purified-2,000-fold from HeLa cells. The factor also increases the unscheduled DNA synthesis of XP-C fibroblasts in vivo after microinjection. Hydrodynamic measurements show that the XP-C complementing factor has a native molecular mass of "160 kDa. The factor binds tightly to single-stranded DNA cellulose, eluting in-1.3 M NaCl. No incision or ATPase activity of the protein alone was detected. XP-C protein is involved in an early stage of repair since its presence was required before the start of gap-filling repair synthesis. In vitro complementation was achieved with naked DNA substrates, and so a primary role in processing chromatin to allow access for repair enzymes seems unlikely. Surprisingly, however, extracts from an XP-C cell line introduced some incisions in W-irradiated DNA; these were unstable in cell extracts and did not lead to complete repair. The data can be explained by a model in which XP-C factor participates in forming one of the repair incisions flanking DNA damage but not the other. In transcribed DNA, its role is subsumed by RNA polymerase and/or transcription coupling factors. Nucleotide excision repair is the main pathway that cells use to remove damage caused to DNA by W light and many chemical mutagens. In humans, a deficiency in this process is associated with the hereditary skin cancer-prone disorder xeroderma pigmentosum (XP).' Of the eight X P complementation groups, group C is one of the most common forms (1). Skin fibroblasts from XP-C cells are hypersensitive to UV light and are greatly, but not completely, defective in nucleotide excision repair. Measurements of cellular repair synthesis show that XP-C cells have 10-20% of the repair synthesis displayed by normal cells. This residual repair synthesis has been the subject of much interest. It arises because XP-C cells repair UV-induced pyrimidine dimers in limited domains (2), even though the cells are unable to remove pyrimidine dimers from most of the genome. The residual repair is strongly associated with transcriptionally active DNA (31, and repair of the tran-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Cell Reports, 2018
Highlights d BRCA2 binds RNAPII to regulate promoter-proximal pausing via PAF1 recruitment d R-lo... more Highlights d BRCA2 binds RNAPII to regulate promoter-proximal pausing via PAF1 recruitment d R-loops accrue at PPP sites after BRCA2 inactivation, causing DNA breakage by ERCC4 d PAF1 depletion phenocopies, while PAF1 overexpression ameliorates, BRCA2 deficiency d Thus, BRCA2 inactivation induces widespread DNA damage via defective RNAPII control
Nucleic acids research, Jan 5, 2016
Homologous DNA recombination (HR) by the RAD51 recombinase enables error-free DNA break repair. T... more Homologous DNA recombination (HR) by the RAD51 recombinase enables error-free DNA break repair. To execute HR, RAD51 first forms a presynaptic filament on single-stranded (ss) DNA, which catalyses pairing with homologous double-stranded (ds) DNA. Here, we report a structure for the presynaptic human RAD51 filament at 3.5-5.0Å resolution using electron cryo-microscopy. RAD51 encases ssDNA in a helical filament of 103Å pitch, comprising 6.4 protomers per turn, with a rise of 16.1Å and a twist of 56.2°. Inter-protomer distance correlates with rotation of an α-helical region in the core catalytic domain that is juxtaposed to ssDNA, suggesting how the RAD51-DNA interaction modulates protomer spacing and filament pitch. We map Fanconi anaemia-like disease-associated RAD51 mutations, clarifying potential phenotypes. We predict binding sites on the presynaptic filament for two modules present in each BRC repeat of the BRCA2 tumour suppressor, a critical HR mediator. Structural modelling sug...
DNA Repair Protocols
Page 1. Dual-Incision Assay 373 30 373 From: Methods in Molecular Biology, Vol. 113: DNA Repair P... more Page 1. Dual-Incision Assay 373 30 373 From: Methods in Molecular Biology, Vol. 113: DNA Repair Protocols: Eukaryotic Systems Edited by: DS Henderson © Humana Press Inc., Totowa, NJ Dual-Incision Assays for Nucleotide ...
Methods in molecular biology (Clifton, N.J.), 2006
Analysis of the mechanism of nucleotide excision repair (NER) using cell-free extract systems and... more Analysis of the mechanism of nucleotide excision repair (NER) using cell-free extract systems and purified proteins requires DNA substrates containing chemically defined lesions that are placed at a unique site in a DNA duplex. In this way, NER can be readily specifically measured by detecting the 24-32 nucleotide products of the dual-incision reaction. This chapter describes several methods for detection of repair of a specific lesion in closed-circular DNA. As a model lesion, we use the well-repaired 1,3-intrastrand d(GpTpG)-cisplatin crosslink. Three methods are given for analysis of repair. One is to incorporate a radioactive label internally near the lesion and measure excision by detecting radioactive excised oligomers. Two other methods use DNA that is not internally labeled so that it can be stored and used when convenient. The first method for detection of repair of such unlabeled DNA is to detect excision products with a labeled complementary oligonucleotide by Southern bl...
The EMBO Journal, 2003
To clarify RAD51 interactions controlling homologous recombination, we report here the crystal st... more To clarify RAD51 interactions controlling homologous recombination, we report here the crystal structure of the full-length RAD51 homolog from Pyrococcus furiosus. The structure reveals how RAD51 proteins assemble into inactive heptameric rings and active DNA-bound ®laments matching three-dimensional electron microscopy reconstructions. A polymerization motif (RAD51-PM) tethers individual subunits together to form assemblies. Subunit interactions support an allosteric`switch' promoting ATPase activity and DNA binding roles for the N-terminal domain helix±hairpin±helix (HhH) motif. Structural and mutational results characterize RAD51 interactions with the breast cancer susceptibility protein BRCA2 in higher eukaryotes. A designed P.furiosus RAD51 mutant binds BRC repeats and forms BRCA2-dependent nuclear foci in human cells in response to g-irradiation-induced DNA damage, similar to human RAD51. These results show that BRCA2 repeats mimic the RAD51-PM and imply analogous RAD51 interactions with RAD52 and RAD54. Both BRCA2 and RAD54 may act as antagonists and chaperones for RAD51 ®lament assembly by coupling RAD51 interface exchanges with DNA binding. Together, these structural and mutational results support an interface exchange hypothesis for coordinated protein interactions in homologous recombination.
Science, 1995
the stimulus velocity was constant, small variations in the edge velocity may have been perceived... more the stimulus velocity was constant, small variations in the edge velocity may have been perceived by the animal because of the curvature of the eye. 18. The value of ox, when measured in conditions of translating and looming stimuli, ranged between 2.9 and 15 rad 1 for angular velocities between 314 and 625 degrees s 1 in different animals. Single exponential fits were consistently better than linear fits; determination coefficients (r2) are 0.962 and 0.935 for exponential fits for Figs. 2E (625 degrees s'1) and 2F (-625 degrees s-1) versus 0.850 and 0.835 for linear fits, respectively. 19. M. O'Shea and C. H. Rowell, J. Exp. Biol. 65, 289 (1976). 20. The exact value of C is unimportant; it is not the absolute firing rate that matters, but the fact that the firing rate peaks and then decreases. 21. The function f (t) will peak when its time derivative is 0. From Eq. 1, df/dt = 0 if 0(tpeak-) =i02(tpeak-) From simple geometrical considerations (Fig. 1 A), 0(t) Sob v)/(d2 + S2Ob) and H(t) = (2Sobj V2 d(d2 +
Proceedings of the National Academy of Sciences, 2009
The breast and ovarian cancer suppressor BRCA2 controls the enzyme RAD51 during homologous DNA re... more The breast and ovarian cancer suppressor BRCA2 controls the enzyme RAD51 during homologous DNA recombination (HDR) to preserve genome stability. BRCA2 binds to RAD51 through 8 conserved BRC repeat motifs dispersed in an 1127-residue region (BRCA2 [BRC1–8] ). Here, we show that BRCA2 [BRC1–8] exerts opposing effects on the binding of RAD51 to single-stranded (ss) versus double-stranded (ds) DNA substrates, enhancing strand exchange. BRCA2 [BRC1–8] alters the electrophoretic mobility of RAD51 bound to an ssDNA substrate, accompanied by an increase in ssDNA-bound protein assemblies, revealed by electron microscopy. Single-molecule fluorescence spectroscopy shows that BRCA2 [BRC1–8] promotes RAD51 loading onto ssDNA. In contrast, BRCA2 [BRC1–8] has a different effect on RAD51 assembly on dsDNA; it suppresses and slows this process. When homologous ssDNA and dsDNA are both present, BRCA2 [BRC1–8] stimulates strand exchange, with delayed RAD51 loading onto dsDNA accompanying the appearanc...
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Papers by Mahmud K . K . Shivji