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At least 91 records · Page 5
All sourcesNASA NTRSDOE OSTI
DOE OSTI2019

The crystal structure of dGTPase reveals the molecular basis of dGTP selectivity

Deoxynucleotide triphosphohydrolases (dNTPases) play a critical role in cellular survival and DNA replication through the proper maintenance of cellular dNTP pools. While the vast majority of these enzymes display broad activity toward canonical dNTPs, such as the dNTPase SAMHD1 that blocks reverse transcription of retroviruses in macrophages by maintaining dNTP pools at low levels, Escherichia coli ( Ec )-dGTPase is the only known enzyme that specifically hydrolyzes dGTP. However, the mechanism behind dGTP selectivity is unclear. Here we present the free-, ligand (dGTP)- and inhibitor (GTP)-bound structures of hexameric Ec- dGTPase, including an X-ray free-electron laser structure of the free Ec- -dGTPase enzyme to 3.2 Å. To obtain this structure, we developed a method that applied UV-fluorescence microscopy, video analysis, and highly automated goniometer-based instrumentation to map and rapidly position individual crystals randomly located on fixed target holders, resulting in the highest indexing rates observed for a serial femtosecond crystallography experiment. Our structures show a highly dynamic active site where conformational changes are coupled to substrate (dGTP), but not inhibitor binding, since GTP locks dGTPase in its apo- form. Moreover, despite no sequence homology, Ec- dGTPase and SAMHD1 share similar active-site and HD motif architectures; however, Ec- dGTPase residues at the end of the substrate-binding pocket mimic Watson–Crick interactions providing guanine base specificity, while a 7-Å cleft separates SAMHD1 residues from dNTP bases, abolishing nucleotide-type discrimination. Furthermore, the structures shed light on the mechanism by which long distance binding (25 Å) of single-stranded DNA in an allosteric site primes the active site by conformationally “opening” a tyrosine gate allowing enhanced substrate binding.

Barnes, Christopher O.↗
NASA NTRS1993

Monoclonal Antibody Testing for Cancer Metastasis

Malignant cells are characterized by the ability to invade surrounding normal tissues. Tumor invasion is abetted by proteolytic enzymes that have been correlated with recurrent disease and metastasis. These enzymes are involved in a cascade of proteolytic interactions with other enzymes and inhibitors which allow cancer cells to dissolve surrounding extracellular matrix, thereby enabling the cells to rapidly invade adjacent tissues and migrate to metastatic sites distant from the primary tumor. Among these proteases are the plasminogen activators (PA), collagenase IV, faminase, and in some cases cathepsin D, which together mediate key steps in the invasion process of metastasis. Cells which have the selective advantage for invasion and metastasis are those capable of regulating their proteolytic activity and proliferation. Cells in the process of invasion would be probably down-regulated for proliferation, but subsequent to attachment and adhesion at a distant site, would then be in a proliferative mode, up-regulating DNA replication. Urokinase (uPA) can be present in the tissues in several molecular forms. The inactive proenzyme is a single chain protein (scuPA) that is cleaved at Lys. 158 to form the double chain, high molecular weight active form (HMW-uPA) of 54 kD. A low molecular weight form (LMW-uPA) can also be produced by cleavage of the HMW-U PA at Lys. 135 - Lys. 136 giving a 35 kD active enzyme. Recently, it has been shown that the HMW active form of urokinase, bound to the tumor cell membrane, is responsible for the local lysis of the extracellular matrix, hence the tissue invasion mechanism for metastasis (Andreasen et al, 19861. Receptor- (membrane) bound uPA is twice as efficient (catalytically) as free fluid-phase uPA. Tho unbound uPA and the LMW form is not responsible for most of the local dissolution of extracellular matrix in the immediate vicinity of the metastatic tumor cell. High levels of urokinase (greater than 3.49 ng/mg of total protein) extracted from breast tumor tissues have recently been shown, together with plasminogen activator inhibitor 1 (PAI-1), to be a good prognostic indicator for high risk of recurrence and shorter patient survival times. In this project, we have attempted to develop immunocytochemical methodologies for the clinical assessment of the expression of urokinase plasminogen activator, which has been implicated to be important for initial steps in tumor invasion, and to relate it to cell proliferation and DNA replication at the single-cell level.

Source record↗
DOE OSTI2018

Escherichia coli single-stranded DNA binding protein SSB promotes AlkB-mediated DNA dealkylation repair

Highlights: • E. coli single stranded DNA binding protein (SSB) wraps single-stranded DNA (ssDNA). • SSB is known to be involved in base excision repair and recombination repair but not in DNA dealkylation repair. • The alkyl-adduct positioned at the 5′ of ssDNA is more accessible to AlkB compared to the internally located alkyl-adducts. • The presence of SSB lowers the K{sub M} of AlkB-catalyzed reaction and promote repair of the longer substrate. • AlkB-mediated repair of methyl adducts from very short ssDNA is rather efficient and SSB-independent. Repair of alkylation damage in DNA is essential for maintaining genome integrity. Escherichia coli (E.coli) protein AlkB removes various alkyl DNA adducts including N1-methyladenine (N{sup 1}meA) and N3-methylcytosine (N{sup 3}meC) by oxidative demethylation. Previous studies showed that AlkB preferentially removes N{sup 1}meA and N{sup 3}meC from single-stranded DNA (ssDNA). It can also remove N{sup 1}meA and N{sup 3}meC from double-stranded DNA by base-flipping. Notably, ssDNA produced during DNA replication and recombination, remains bound to E. coli single-stranded DNA binding protein SSB and it is not known whether AlkB can repair methyl adduct present in SSB-coated DNA. Here we have studied AlkB-mediated DNA repair using SSB-bound DNA as substrate. In vitro repair reaction revealed that AlkB could efficiently remove N{sup 3}meC adducts inasmuch as DNA length is shorter than 20 nucleotides. However, when longer N{sup 3}meC-containing oligonuleotides were used as the substrate, efficiency of AlkB catalyzed reaction was abated compared to SSB-bound DNA substrate of identical length. Truncated SSB containing only the DNA binding domain could also support the stimulation of AlkB activity, suggesting the importance of SSB-DNA interaction for AlkB function. Using 70-mer oligonucleotide containing single N{sup 3}meC we demonstrate that SSB-AlkB interaction promotes faster repair of the methyl DNA adducts.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2019

Two-tiered enforcement of high-fidelity DNA ligation

DNA ligases catalyze the joining of DNA strands to complete DNA replication, recombination and repair transactions. To protect the integrity of the genome, DNA ligase 1 (LIG1) discriminates against DNA junctions harboring mutagenic 3'-DNA mismatches or oxidative DNA damage, but how such high-fidelity ligation is enforced is unknown. Here, X-ray structures and kinetic analyses of LIG1 complexes with undamaged and oxidatively damaged DNA unveil that LIG1 employs Mg 2+ -reinforced DNA binding to validate DNA base pairing during the adenylyl transfer and nick-sealing ligation reaction steps. Our results support a model whereby LIG1 fidelity is governed by a high-fidelity (HiFi) interface between LIG1, Mg 2+ , and the DNA substrate that tunes the enzyme to release pro-mutagenic DNA nicks. In a second tier of protection, LIG1 activity is surveilled by Aprataxin (APTX), which suppresses mutagenic and abortive ligation at sites of oxidative DNA damage.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Poxvirus uracil‐DNA glycosylase—An unusual member of the family I uracil‐DNA glycosylases

Abstract Uracil‐DNA glycosylases are ubiquitous enzymes, which play a key role repairing damages in DNA and in maintaining genomic integrity by catalyzing the first step in the base excision repair pathway. Within the superfamily of uracil‐DNA glycosylases family I enzymes or UNGs are specific for recognizing and removing uracil from DNA. These enzymes feature conserved structural folds, active site residues and use common motifs for DNA binding, uracil recognition and catalysis. Within this family the enzymes of poxviruses are unique and most remarkable in terms of amino acid sequences, characteristic motifs and more importantly for their novel non‐enzymatic function in DNA replication. UNG of vaccinia virus, also known as D4, is the most extensively characterized UNG of the poxvirus family. D4 forms an unusual heterodimeric processivity factor by attaching to a poxvirus‐specific protein A20, which also binds to the DNA polymerase E9 and recruits other proteins necessary for replication. D4 is thus integrated in the DNA polymerase complex, and its DNA‐binding and DNA scanning abilities couple DNA processivity and DNA base excision repair at the replication fork. The adaptations necessary for taking on the new function are reflected in the amino acid sequence and the three‐dimensional structure of D4. An overview of the current state of the knowledge on the structure‐function relationship of D4 is provided here.

Schormann, Norbert↗
DOE OSTI2015

Proteasome activity is important for replication recovery, CHK1 phosphorylation and prevention of G2 arrest after low-dose formaldehyde

Formaldehyde (FA) is a human carcinogen with numerous sources of environmental and occupational exposures. This reactive aldehyde is also produced endogenously during metabolism of drugs and other processes. DNA–protein crosslinks (DPCs) are considered to be the main genotoxic lesions for FA. Accumulating evidence suggests that DPC repair in high eukaryotes involves proteolysis of crosslinked proteins. Here, we examined a role of the main cellular proteolytic machinery proteasomes in toxic responses of human lung cells to low FA doses. We found that transient inhibition of proteasome activity increased cytotoxicity and diminished clonogenic viability of FA-treated cells. Proteasome inactivation exacerbated suppressive effects of FA on DNA replication and increased the levels of the genotoxic stress marker γ-H2AX in normal human cells. A transient loss of proteasome activity in FA-exposed cells also caused delayed perturbations of cell cycle, which included G2 arrest and a depletion of S-phase populations at FA doses that had no effects in control cells. Proteasome activity diminished p53-Ser15 phosphorylation but was important for FA-induced CHK1 phosphorylation, which is a biochemical marker of DPC proteolysis in replicating cells. Unlike FA, proteasome inhibition had no effect on cell survival and CHK1 phosphorylation by the non-DPC replication stressor hydroxyurea. Overall, we obtained evidence for the importance of proteasomes in protection of human cells against biologically relevant doses of FA. Biochemically, our findings indicate the involvement of proteasomes in proteolytic repair of DPC, which removes replication blockage by these highly bulky lesions. - Highlights: • Proteasome inhibition enhances cytotoxicity of low-dose FA in human lung cells. • Active proteasomes diminish replication-inhibiting effects of FA. • Proteasome activity prevents delayed G2 arrest in FA-treated cells. • Proteasome inhibition exacerbates replication stress by FA in normal human cells. • Protective role of proteasomes is linked to repair of DNA–protein crosslinks.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Dual role of Fam208a during zygotic cleavage and early embryonic development

Maintenance of genome stability is essential for every living cell as genetic information is repeatedly challenged during DNA replication in each cell division event. Errors, defects, delays, and mistakes that arise during mitosis or meiosis lead to an activation of DNA repair processes and in case of their failure, programmed cell death, i.e. apoptosis, could be initiated. Fam208a is a protein whose importance in heterochromatin maintenance has been described recently. In this work, we describe the crucial role of Fam208a in sustaining genome stability during cellular division. }The targeted depletion of Fam208a in mice using CRISPR/Cas9 led to embryonic lethality before E12.5. We also used the siRNA approach to downregulate Fam208a in zygotes to avoid the influence of maternal RNA in the early stages of development. This early downregulation increased arresting of the embryonal development at the two-cell stage and the occurrence of multipolar spindles formation. To investigate this further, we used the yeast two-hybrid (Y2H) system and identified new putative interaction partners Gpsm2, Svil, and Itgb3bp. Their co-expression with Fam208a was assessed by RT-qPCR profiling and in situ hybridization [1] in multiple murine tissues. Based on these results we proposed that Fam208a functions within the HUSH complex by interaction with Mphosph8 as these proteins are not only able to physically interact but also co-localise. We are bringing new evidence that Fam208a is a multi-interacting protein affecting genome stability on the cell division level at the earliest stages of development and by interaction with methylation complex in adult tissues. }In addition to its epigenetic functions, Fam208a appears to have an important role in the zygotic division, possibly via interaction with newly identified putative partners Gpsm2, Svil, and Itgb3bp.

60 APPLIED LIFE SCIENCES↗
NASA NTRS1992

Crystal structure of an Okazaki fragment at 2-A resolution

In DNA replication, Okazaki fragments are formed as double-stranded intermediates during synthesis of the lagging strand. They are composed of the growing DNA strand primed by RNA and the template strand. The DNA oligonucleotide d(GGGTATACGC) and the chimeric RNA-DNA oligonucleotide r(GCG)d(TATACCC) were combined to form a synthetic Okazaki fragment and its three-dimensional structure was determined by x-ray crystallography. The fragment adopts an overall A-type conformation with 11 residues per turn. Although the base-pair geometry, particularly in the central TATA part, is distorted, there is no evidence for a transition from the A- to the B-type conformation at the junction between RNA.DNA hybrid and DNA duplex. The RNA trimer may, therefore, lock the complete fragment in an A-type conformation.

Non-NASA Center↗
DOE OSTI2017

A Specific Mutational Signature Associated with DNA 8-Oxoguanine Persistence in MUTYH-defective Colorectal Cancer

8-Oxoguanine, a common mutagenic DNA lesion, generates G:C > T:A transversions via mispairing with adenine during DNA replication. When operating normally, the MUTYH DNA glycosylase prevents 8-oxoguanine-related mutagenesis by excising the incorporated adenine. Biallelic MUTYH mutations impair this enzymatic function and are associated with colorectal cancer (CRC) in MUTYH-Associated Polyposis (MAP) syndrome. Here in this paper, we perform whole-exome sequencing that reveals a modest mutator phenotype in MAP CRCs compared to sporadic CRC stem cell lines or bulk tumours. The excess G:C > T:A transversion mutations in MAP CRCs exhibits a novel mutational signature, termed Signature 36, with a strong sequence dependence. The MUTYH mutational signature reflecting persistent 8-oxoG:A mismatches occurs frequently in the APC, KRAS, PIK3CA, FAT4, TP53, FAT1, AMER1, KDM6A, SMAD4 and SMAD2 genes that are associated with CRC. In conclusion, the occurrence of Signature 36 in other types of human cancer indicates that DNA 8-oxoguanine-related mutations might contribute to the development of cancer in other organs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Structures of ATP-bound DNA ligase D in a closed domain conformation reveal a network of amino acid and metal contacts to the ATP phosphates

DNA ligases are the sine qua non of genome integrity and essential for DNA replication and repair in all organisms. DNA ligases join 3'-OH and 5'-PO 4 ends via a series of three nucleotidyl transfer steps. In step 1, ligase reacts with ATP or NAD + to form a covalent ligase-(lysyl-N$ζ$)–AMP intermediate and release pyrophosphate (PP i ) or nicotinamide mononucleotide. In step 2, AMP is transferred from ligase-adenylate to the 5'-PO 4 DNA end to form a DNA-adenylate intermediate (AppDNA). In step 3, ligase catalyzes attack by a DNA 3'-OH on the DNA-adenylate to seal the two ends via a phosphodiester bond and release AMP. Eukaryal, archaeal, and many bacterial and viral DNA ligases are ATP-dependent. The catalytic core of ATP-dependent DNA ligases consists of an N-terminal nucleotidyltransferase domain fused to a C-terminal OB domain. Here we report crystal structures at 1.4–1.8 Å resolution of Mycobacterium tuberculosis LigD, an ATP-dependent DNA ligase dedicated to nonhomologous end joining, in complexes with ATP that highlight large movements of the OB domain (~50 Å), from a closed conformation in the ATP complex to an open conformation in the covalent ligase-AMP intermediate. The LigD·ATP structures revealed a network of amino acid contacts to the ATP phosphates that stabilize the transition state and orient the PP i leaving group. A complex with ATP and magnesium suggested a two-metal mechanism of lysine adenylylation driven by a catalytic Mg 2+ that engages the ATP $α$ phosphate and a second metal that bridges the ATP $β$ and $γ$ phosphates.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Nonenzymatic Role for WRN in Preserving Nascent DNA Strands after Replication Stress

WRN, the protein defective in Werner syndrome (WS), is a multifunctional nuclease involved in DNA damage repair, replication, and genome stability maintenance. It was assumed that the nuclease activities of WRN were critical for these functions. Here, we report a nonenzymatic role for WRN in preserving nascent DNA strands following replication stress. We found that lack of WRN led to shortening of nascent DNA strands after replication stress. Furthermore, we discovered that the exonuclease activity of MRE11 was responsible for the shortening of newly replicated DNA in the absence of WRN. Mechanistically, the N-terminal FHA domain of NBS1 recruits WRN to replication-associated DNA double-stranded breaks to stabilize Rad51 and to limit the nuclease activity of its C-terminal binding partner MRE11. Thus, this previously unrecognized nonenzymatic function of WRN in the stabilization of nascent DNA strands sheds light on the molecular reason for the origin of genome instability in WS individuals.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Mechanistic Basis for the Bypass of a Bulky DNA Adduct Catalyzed by a Y-Family DNA Polymerase

1-Nitropyrene (1-NP), an environmental pollutant, induces DNA damage in vivo and is considered to be carcinogenic. The DNA adducts formed by the 1-NP metabolites stall replicative DNA polymerases but are presumably bypassed by error-prone Y-family DNA polymerases at the expense of replication fidelity and efficiency in vivo. Our running start assays confirmed that a site-specifically placed 8-(deoxyguanosin-N 2 -yl)-1-aminopyrene (dG 1,8 ), one of the DNA adducts derived from 1-NP, can be bypassed by Sulfolobus solfataricus DNA polymerase IV (Dpo4), although this representative Y-family enzyme was paused strongly by the lesion. Pre-steady-state kinetic assays were employed to determine the low nucleotide incorporation fidelity and establish a minimal kinetic mechanism for the dG 1,8 bypass by Dpo4. To reveal a structural basis for dCTP incorporation opposite dG 1,8 , we solved the crystal structures of the complexes of Dpo4 and DNA containing a templating dG 1,8 lesion in the absence or presence of dCTP. The Dpo4·DNA-dG 1,8 binary structure shows that the aminopyrene moiety of the lesion stacks against the primer/template junction pair, while its dG moiety projected into the cleft between the Finger and Little Finger domains of Dpo4. In the Dpo4·DNA-dG 1,8 ·dCTP ternary structure, the aminopyrene moiety of the dG 1,8 lesion, is sandwiched between the nascent and junction base pairs, while its base is present in the major groove. Moreover, dCTP forms a Watson–Crick base pair with dG, two nucleotides upstream from the dG 1,8 site, creating a complex for “-2” frameshift mutation. Mechanistically, these crystal structures provide additional insight into the aforementioned minimal kinetic mechanism.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Replication stress induced site-specific phosphorylation targets WRN to the ubiquitin-proteasome pathway

Faithful and complete genome replication in human cells is essential for preventing the accumulation of cancer-promoting mutations. WRN, the protein defective in Werner syndrome, plays critical roles in preventing replication stress, chromosome instability, and tumorigenesis. Herein, we report that ATR-mediated WRN phosphorylation is needed for DNA replication and repair upon replication stress. A serine residue, S1141, in WRN is phosphorylated in vivo by the ATR kinase in response to replication stress. ATR-mediated WRN S1141 phosphorylation leads to ubiquitination of WRN, facilitating the reversible interaction of WRN with perturbed replication forks and subsequent degradation of WRN. The dynamic interaction between WRN and DNA is required for the suppression of new origin firing and Rad51-dependent double-stranded DNA break repair. Significantly, ATR-mediated WRN phosphorylation is critical for the suppression of chromosome breakage during replication stress. These findings reveal a unique role for WRN as a modulator of DNA repair, replication, and recombination, and link ATR-WRN signaling to the maintenance of genome stability.

Cell Biology↗
DOE OSTI2016

Dietary flavonoid derivatives enhance chemotherapeutic effect by inhibiting the DNA damage response pathway

Flavonoids are the most common group of polyphenolic compounds and abundant in dietary fruits and vegetables. Diet high in vegetables or dietary flavonoid supplements is associated with reduced mortality rate for patients with breast cancer. Many studies have been proposed for mechanisms linking flavonoids to improving chemotherapy efficacy in many types of cancers, but data on this issue is still limited. Herein, we report on a new mechanism through which dietary flavonoids inhibit DNA damage checkpoints and repair pathways. We found that dietary flavonoids could inhibit Chk1 phosphorylation and decrease clonogenic cell growth once breast cancer cells receive ultraviolet irradiation, cisplatin, or etoposide treatment. Since the ATR-Chk1 pathway mainly involves response to DNA replication stress, we propose that flavonoid derivatives reduce the side effect of chemotherapy by improving the sensitivity of cycling cells. Therefore, we propose that increasing intake of common dietary flavonoids is beneficial to breast cancer patients who are receiving DNA-damaging chemotherapy, such as cisplatin or etoposide-based therapy. - Highlights: • First report on inhibition of both DNA damage and repair by dietary flavonoids • Dietary flavonoids inhibit cisplatin- and UV-induced Chk1 phosphorylation. • Flavonoids combined with cisplatin or UV treatment show notable growth inhibition. • Promising treatment proposal for patients who are receiving adjuvant chemotherapy.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

In vitro reconstitution and biochemical analyses of the Schizosaccharomyces pombe nucleosome

Schizosaccharomyces pombe, which has a small genome but shares many physiological functions with higher eukaryotes, is a useful single-cell, model eukaryotic organism. In particular, many features concerning chromatin structure and dynamics, including heterochromatin, centromeres, telomeres, and DNA replication origins, are well conserved between S. pombe and higher eukaryotes. However, the S. pombe nucleosome, the fundamental structural unit of chromatin, has not been reconstituted in vitro. In the present study, we established the method to purify S. pombe histones H2A, H2B, H3, and H4, and successfully reconstituted the S. pombe nucleosome in vitro. Our thermal stability assay and micrococcal nuclease treatment assay revealed that the S. pombe nucleosome is markedly unstable and its DNA ends are quite accessible, as compared to the canonical human nucleosome. These findings are important to understand the mechanisms of epigenetic genomic DNA regulation in fission yeast. - Highlights: • S. pombe histones were purified as recombinant proteins. • The recombinant S. pombe histones efficiently form nucleosomes in vitro. • The S. pombe nucleosome has distinct stability and DNA dynamics.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

The human CTC1/STN1/TEN1 complex regulates telomere maintenance in ALT cancer cells

Maintaining functional telomeres is important for long-term proliferation of cells. About 15% of cancer cells are telomerase-negative and activate the alternative-lengthening of telomeres (ALT) pathway to maintain their telomeres. Recent studies have shown that the human CTC1/STN1/TEN1 complex (CST) plays a multi-faceted role in telomere maintenance in telomerase-expressing cancer cells. However, the role of CST in telomere maintenance in ALT cells is unclear. Here, we report that human CST forms a functional complex localizing in the ALT-associated PML bodies (APBs) in ALT cells throughout the cell cycle. Suppression of CST induces telomere instabilities including telomere fragility and elevates telomeric DNA recombination, leading to telomere dysfunction. In addition, CST deficiency significantly diminishes the abundance of extrachromosomal circular telomere DNA known as C-circles and t-circles. Suppression of CST also results in multinucleation in ALT cells and impairs cell proliferation. Our findings imply that the CST complex plays an important role in regulating telomere maintenance in ALT cells. - Highlights: • CST localizes at telomeres and ALT-associated PML bodies in ALT cells throughout the cell cycle. • CST is important for promoting telomeric DNA replication in ALT cells. • CST deficiency decreases ECTR formation and increases T-SCE. • CST deficiency impairs ALT cell proliferation and results in multinucleation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

p53R2 regulates thioredoxin reductase activity through interaction with TrxR2

Ribonucleotide reductase small subunit p53R2 is a member of the ribonucleotide reductase family that supplies dNTPs for nuclear and mitochondrial DNA replication and repair. Here, we have identified a mitochondrial thioredoxin reductase 2 (TrxR2) as a novel p53R2-binding protein. We demonstrated a direct interaction between the two, and observed that p53R2 stimulated the enzymatic activity of TrxR in vitro. Moreover, TrxR2 activity was significantly lower in p53R2 knockdown cells, and increased when p53R2 was overexpressed, effects that were independent of p53. Furthermore, p53R2 knockdown suppressed UV-induced TrxR activity. These findings suggest that p53R2 acts as a positive regulator of TrxR2 activity in mitochondria both under normal physiological conditions and during the cellular response to DNA damage. - Highlights: • P53R2 binds to thioredoxin reductase 2 (TrxR2) in vivo and in vitro. • P53R2 enhances TrxR activity. • P53R2 regulates TrxR activity in a p53-independent manner. • P53R2 acts as a positive regulator of TrxR2 activity under both normal and DNA damage conditions.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Crystal structure of the histone heterodimer containing histone variant H2A.Bbd

H2A.Bbd, the most divergent histone variant among all known H2A type histones, is involved in gene transcription, spermiogenesis, DNA replication and RNA splicing. Incorporation of H2A.Bbd-H2B dimer, a fundamental unit of H2A.Bbd nucleosome, modulate structures of nucleosome or chromatin, but the underlying mechanism remains elusive. Here we determined a crystal structure of H2A.Bbd-H2B dimer at 2.6 Å resolution. Although the H2A.Bbd-H2B dimer structure largely resembles that of H2A-H2B, substitution of H2A αC helix residues by H2A.Bbd counterparts lead to the transition of a long αC-helix to the short 3{sub 10}-helix, likely owing to the rearrangement of the hydrogen-bond network. Moreover, structural comparison revealed a strikingly altered electrostatic potential surface for H2A.Bbd-H2B dimer displaying a diminished DNA binding capability. Our study provides the first high-resolution structure of histone variant H2A.Bbd and shed a light on biological function of H2A.Bbd.

60 APPLIED LIFE SCIENCES↗