ORCID Profile
0000-0001-5581-4616
Current Organisations
National Institutes of Health
,
King Abdullah International Medical Research Center
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Publisher: American Chemical Society (ACS)
Date: 12-04-2016
Publisher: Elsevier BV
Date: 02-1999
Abstract: The UmuD-like proteins are best characterized for their role in damage-induced SOS mutagenesis. An essential step in this process is the enzymatic self-processing of the UmuD-like proteins. This reaction is thought to occur either via an intramolecular or intermolecular self-cleavage mechanism. Here, we demonstrate that it can also occur via an heterologous intermolecular cleavage reaction. The Escherichia coli UmuD enzyme demonstrated the broadest substrate specificity, cleaving both E. coli and Salmonella typhimurium UmuD substrates in vivo. In comparison, the wild-type S. typhimurium UmuD (UmuDSt) and MucA enzymes catalyzed intermolecular self-cleavage, but did not facilitate heterologous cleavage. Heterologous cleavage by the UmuDSt enzyme was, however, observed with chimeric UmuD substrates that possess residues 30-55 of UmuDSt. We have further localized the residue predominantly responsible for UmuDSt-catalyzed heterologous cleavage to Ser50 in the substrate molecule. We hypothesize that changes at this residue affect the positioning of the cleavage site of a substrate molecule within the catalytic cleft of the UmuDSt enzyme by affecting the formation of a so-called UmuD "filament-dimer". This hypothesis is further supported by the observation that mutations known to disrupt an E. coli UmuD' filament dimer also block intermolecular UmuDEc cleavage.
Publisher: Cold Spring Harbor Laboratory
Date: 10-09-2018
DOI: 10.1101/413880
Abstract: The RecA protein orchestrates the cellular response to DNA damage via its multiple roles in the bacterial SOS response. Lack of tools that provide unambiguous access to the various RecA states within the cell have prevented understanding of the spatial and temporal changes in RecA structure/function that underlie control of the damage response. Here, we develop a monomeric C-terminal fragment of the λ repressor as a novel fluorescent probe that specifically interacts with RecA filaments on single-stranded DNA (RecA*). Single-molecule imaging techniques in live cells demonstrate that RecA is largely sequestered in storage structures during normal metabolism. Upon DNA damage, the storage structures dissolve and the cytosolic pool of RecA rapidly nucleates to form early SOS-signaling complexes, maturing into DNA-bound RecA bundles at later time points. Both before and after SOS induction, RecA* largely appears at locations distal from replisomes. Upon completion of repair, RecA storage structures reform.
Publisher: eLife Sciences Publications, Ltd
Date: 05-02-2019
DOI: 10.7554/ELIFE.42761
Abstract: The RecA protein orchestrates the cellular response to DNA damage via its multiple roles in the bacterial SOS response. Lack of tools that provide unambiguous access to the various RecA states within the cell have prevented understanding of the spatial and temporal changes in RecA structure/function that underlie control of the damage response. Here, we develop a monomeric C-terminal fragment of the λ repressor as a novel fluorescent probe that specifically interacts with RecA filaments on single-stranded DNA (RecA*). Single-molecule imaging techniques in live cells demonstrate that RecA is largely sequestered in storage structures during normal metabolism. Upon DNA damage, the storage structures dissolve and the cytosolic pool of RecA rapidly nucleates to form early SOS-signaling complexes, maturing into DNA-bound RecA bundles at later time points. Both before and after SOS induction, RecA* largely appears at locations distal from replisomes. Upon completion of repair, RecA storage structures reform.
Publisher: MDPI AG
Date: 02-05-2020
DOI: 10.3390/V12050502
Abstract: The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a lethal zoonotic pathogen circulating in the Arabian Peninsula since 2012. There is no vaccine for MERS and anti-viral treatment is generally not applicable. We investigated the evolution of the MERS-CoV spike gene sequences and changes in viral loads over time from patients in Saudi Arabia from 2015–2017. All the MERS-CoV strains belonged to lineage 5, and showed high sequence homology (99.9%) to 2017 strains. Recombination analysis showed a potential recombination event in study strains from patients in Saudi Arabia. The spike gene showed eight amino acid substitutions, especially between the A1 and B5 lineage, and contained positively selected codon 1020. We also determined that the viral loads were significantly (p 0.001) higher in fatal cases, and virus shedding was prolonged in some fatal cases beyond 21 days. The viral concentration peaked during the first week of illness, and the lower respiratory specimens had higher levels of MERS-CoV RNA. The presence of the ersifying selection and the topologies with the structural mapping of residues under purifying selection suggested that codon 1020 might have a role in the evolution of spike gene during the ergence of different lineages. This study will improve our understanding of the evolution of MERS-CoV, and also highlights the need for enhanced surveillance in humans and dromedaries. The presence of amino acid changes at the N-terminal domain and structural mapping of residues under positive selection at heptad repeat 1 provides better insight into the adaptive evolution of the spike gene and might have a potential role in virus-host tropism and pathogenesis.
Publisher: Elsevier BV
Date: 2021
Publisher: Elsevier BV
Date: 02-2021
Publisher: Oxford University Press (OUP)
Date: 10-02-2202
DOI: 10.1093/NAR/GKAD038
Abstract: In Escherichia coli, replication of both strands of genomic DNA is carried out by a single replicase—DNA polymerase III holoenzyme (pol III HE). However, in certain genetic backgrounds, the low-fidelity TLS polymerase, DNA polymerase V (pol V) gains access to undamaged genomic DNA where it promotes elevated levels of spontaneous mutagenesis preferentially on the lagging strand. We employed active site mutants of pol III (pol IIIα_S759N) and pol V (pol V_Y11A) to analyze ribonucleotide incorporation and removal from the E. coli chromosome on a genome-wide scale under conditions of normal replication, as well as SOS induction. Using a variety of methods tuned to the specific properties of these polymerases (analysis of lacI mutational spectra, lacZ reversion assay, HydEn-seq, alkaline gel electrophoresis), we present evidence that repair of ribonucleotides from both DNA strands in E. coli is unequal. While RNase HII plays a primary role in leading-strand Ribonucleotide Excision Repair (RER), the lagging strand is subject to other repair systems (RNase HI and under conditions of SOS activation also Nucleotide Excision Repair). Importantly, we suggest that RNase HI activity can also influence the repair of single ribonucleotides incorporated by the replicase pol III HE into the lagging strand.
Publisher: MDPI AG
Date: 02-10-2020
Abstract: Many endogenous and exogenous factors can induce genomic instability in human cells, in the form of DNA damage and mutations, that predispose them to cancer development. Normal cells rely on DNA damage bypass pathways such as translesion synthesis (TLS) and template switching (TS) to replicate past lesions that might otherwise result in prolonged replication stress and lethal double-strand breaks (DSBs). However, due to the lower fidelity of the specialized polymerases involved in TLS, the activation and suppression of these pathways must be tightly regulated by post-translational modifications such as ubiquitination in order to limit the risk of mutagenesis. Many cancer cells rely on the deregulation of DNA damage bypass to promote carcinogenesis and tumor formation, often giving them heightened resistance to DNA damage from chemotherapeutic agents. In this review, we discuss the key functions of ubiquitin and ubiquitin-like proteins in regulating DNA damage bypass in human cells, and highlight ways in which these processes are both deregulated in cancer progression and might be targeted in cancer therapy.
Publisher: MDPI AG
Date: 19-01-2021
DOI: 10.3390/V13010136
Abstract: In December 2019, the emergence of SARS-CoV-2 virus in China led to a pandemic. Since both Influenza Like Illness (ILI) and COVID-19 case definitions overlap, we re-investigated the ILI cases using PCR for the presence of SARS-CoV-2 in 739 nasopharyngeal swabs collected from November 2019 to March 2020. SARS-CoV-2 RNA was found in 37 s les (5%) collected mostly during February 2020. It was followed by confirmation of evolutionary and spatial relationships using next generation sequencing (NGS). We observed that the overall incidence of ILI cases during 2019–2020 influenza season was considerably higher than previous years and was gradually replaced with SARS-CoV-2, which indicated a silent transmission among ambulatory patients. Sequencing of representative isolates confirmed independent introductions and silent transmission earlier than previously thought. Evolutionary and spatial analyses revealed clustering in the GH clade, characterized by three amino acid substitutions in spike gene (D614G), RdRp (P323L) and NS3 (Q57H). P323L causes conformational change near nsp8 binding site that might affect virus replication and transcription. In conclusion, assessment of the community transmission among patients with mild COVID-19 illness, particularly those without epidemiological link for acquiring the virus, is of utmost importance to guide policy makers to optimize public health interventions. The detection of SARS-CoV-2 in ILI cases shows the importance of ILI surveillance systems and warrants its further strengthening to mitigate the ongoing transmission of SARS-CoV-2. The effect of NS3 substitutions on oligomerization or membrane channel function (intra- and extracellular) needs functional validation.
Publisher: Elsevier BV
Date: 12-2019
Publisher: Frontiers Media SA
Date: 05-08-2020
Publisher: eLife Sciences Publications, Ltd
Date: 08-01-2019
Publisher: Public Library of Science (PLoS)
Date: 19-01-2018
Publisher: Public Library of Science (PLoS)
Date: 28-08-2015
Publisher: Oxford University Press (OUP)
Date: 20-07-2020
DOI: 10.1093/NAR/GKAA597
Abstract: Several functions have been proposed for the Escherichia coli DNA polymerase IV (pol IV). Although much research has focused on a potential role for pol IV in assisting pol III replisomes in the bypass of lesions, pol IV is rarely found at the replication fork in vivo. Pol IV is expressed at increased levels in E. coli cells exposed to exogenous DNA damaging agents, including many commonly used antibiotics. Here we present live-cell single-molecule microscopy measurements indicating that double-strand breaks induced by antibiotics strongly stimulate pol IV activity. Exposure to the antibiotics ciprofloxacin and trimethoprim leads to the formation of double strand breaks in E. coli cells. RecA and pol IV foci increase after treatment and exhibit strong colocalization. The induction of the SOS response, the appearance of RecA foci, the appearance of pol IV foci and RecA-pol IV colocalization are all dependent on RecB function. The positioning of pol IV foci likely reflects a physical interaction with the RecA* nucleoprotein filaments that has been detected previously in vitro. Our observations provide an in vivo substantiation of a direct role for pol IV in double strand break repair in cells treated with double strand break-inducing antibiotics.
Location: No location found
Location: Saudi Arabia
No related grants have been discovered for Roger Woodgate.