Orchestration of base excision repair by controlling the rates of enzymatic activities

Base excision repair (BER) is one of the major pathways for repair of simple DNA base lesions and is carried out through a series of coordinated reactions relying on several different enzymatic activities and accessory proteins. Imbalance of BER activities has been reported to be linked to genetic instability and cancer. To experimentally address the mechanisms orchestrating BER, we monitored both the overall rate and the rate-limiting steps in the repair in cell-free extracts of five different endogenously occurring DNA lesions (abasic site, uracil, 8-oxoguanine, hypoxanthine and 5,6-dihydrouracil) and the effect of addition of rate-limiting BER components on the rate and co-ordination of BER reactions. We find that several mechanisms including regulation of DNA glycosylase turnover and involvement of poly(ADP-ribose) polymerase participate in synchronization of the repair events. We also find that repair of different DNA lesions involves different mechanisms for optimizing repair rates without accumulation of intermediates. Repair of some lesions such as 8-oxoguanine is regulated by glycosylase turnover and progress without substantial accumulation of repair intermediates. However, during repair of the apurinic/apyrimidinic (AP) sites or 5,6-dihydrouracil, poly(ADP-ribose) polymerase plays an important role in the coordination of the rates of repair reactions.     

A Highlights from MBoC Selection: Srv2/cyclase-associated protein forms hexameric shurikens that directly catalyze actin filament severing by cofilin

Actin filament severing is critical for the dynamic turnover of cellular actin networks. Cofilin severs filaments, but additional factors may be required to increase severing efficiency in vivo. Srv2/cyclase-associated protein (CAP) is a widely expressed protein with a role in binding and recycling actin monomers ascribed to domains in its C-terminus (C-Srv2). In this paper, we report a new biochemical and cellular function for Srv2/CAP in directly catalyzing cofilin-mediated severing of filaments. This function is mediated by its N-terminal half (N-Srv2), and is physically and genetically separable from C-Srv2 activities. Using dual-color total internal reflection fluorescence microscopy, we determined that N-Srv2 stimulates filament disassembly by increasing the frequency of cofilin-mediated severing without affecting cofilin binding to filaments. Structural analysis shows that N-Srv2 forms novel hexameric star-shaped structures, and disrupting oligomerization impairs N-Srv2 activities and in vivo function. Further, genetic analysis shows that the combined activities of N-Srv2 and Aip1 are essential in vivo. These observations define a novel mechanism by which the combined activities of cofilin and Srv2/CAP lead to enhanced filament severing and support an emerging view that actin disassembly is controlled not by cofilin alone, but by a more complex set of factors working in concert.     

CK2 phosphorylation of XRCC1 facilitates dissociation from DNA and single-strand break formation during base excision repair

CK2 phosphorylates the scaffold protein XRCC1, which is required for efficient DNA single-strand break (SSB) repair. Here, we express an XRCC1 protein (XRCC1(ckm)) that cannot be phosphorylated by CK2 in XRCC1 mutated EM9 cells and show that the role of this post-translational modification gives distinct phenotypes in SSB repair and base excision repair (BER). Interestingly, we find that fewer SSBs are formed during BER after treatment with the alkylating agent dimethyl sulfate (DMS) in EM9 cells expressing XRCC1(ckm) (CKM cells) or following inhibition with the CK2 inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT). We also show that XRCC1(ckm) protein has a higher affinity for DNA than wild type XRCC1 protein and resides in an immobile fraction on DNA, in particular after damage. We propose a model whereby the increased affinity for DNA sequesters XRCC1(ckm) and the repair enzymes associated with it, at the repair site, which retards kinetics of BER. In conclusion, our results indicate that phosphorylation of XRCC1 by CK2 facilitates the BER incision step, likely by promoting dissociation from DNA.     

Mammalian Base Excision Repair: the Forgotten Archangel

Base excision repair (BER) is a frontline repair system that is responsible for maintaining genome integrity and thus preventing premature aging, cancer and many other human diseases by repairing thousands of DNA lesions and strand breaks continuously caused by endogenous and exogenous mutagens. This fundamental and essential function of BER not only necessitates tight control of the continuous availability of basic components for fast and accurate repair, but also requires temporal and spatial coordination of BER and cell cycle progression to prevent replication of damaged DNA. The major goal of this review is to critically examine controversial and newly emerging questions about mammalian BER pathways, mechanisms regulating BER capacity, BER responses to DNA damage and their links to checkpoint control of DNA replication.     

Redox regulation of morphology,cell stiffness,and lectin-induced aggregation of human platelets

Redox regulation and carbohydrate recognition are potent molecular mechanisms which can contribute to platelet aggregation in response to various stimuli. The purpose of this study is to investigate the relationship between these mechanisms and to examine whether cell surface glycocalyx and cell stiffness of human platelets are sensitive to the redox potential formed by glutathione. To this end, human platelets were treated with different concentrations (0.05 μM to 6 mM) and ratios of reduced or oxidized glutathione (GSH or GSSG), and platelet morphological, mechanical, and functional properties were determined using conventional light microscopy, atomic force microscopy, and lectin-induced cell aggregation analysis. It was found that lowering the glutathione redox potential changed platelet morphology and increased platelet stiffness as well as modulated nonuniformly platelet aggregation in response to plant lectins with different carbohydrate-binding specificity including wheat germ agglutinin, Sambucus nigra agglutinin, and Canavalia ensiformis agglutinin. Extracellular redox potential and redox buffering capacity of the GSSG/2GSH couple were shown to control the availability of specific lectin-binding glycoligands on the cell surface, while the intracellular glutathione redox state affected the general functional ability of platelets to be aggregated independently of the type of lectins. Our data provide the first experimental evidence that glutathione as a redox molecule can affect the mechanical stiffness of human platelets and induce changes of the cell surface glycocalyx, which may represent a new mechanism of redox regulation of intercellular contacts.     

Expression and characterization of the bacterial mechanosensitive channel MscS in Xenopus laevis oocytes

We have successfully expressed and characterized mechanosensitive channel of small conductance (MscS) from Escherichia coli in oocytes of the African clawed frog, Xenopus laevis. MscS expressed in oocytes has the same single-channel conductance and voltage dependence as the channel in its native environment. Two hallmarks of MscS activity, the presence of conducting substates at high potentials and reversible adaptation to a sustained stimulus, are also exhibited by oocyte-expressed MscS. In addition to its ease of use, the oocyte system allows the user to work with relatively large patches, which could be an advantage for the visualization of membrane deformation. Furthermore, MscS can now be compared directly to its eukaryotic homologues or to other mechanosensitive channels that are not easily studied in E. coli.     

A Model of Genetic Search for Beneficial Mutations: Estimating the Constructive Capacities of Mutagenesis

We attempted to answer the following question: What evolutionary conditions are required to generate novel genetic modules? Our broad formulation of the problem allows us to simultaneously consider such issues as the relationship between the stage of "genetic search" and the rate of adaptive evolution; the theoretical limits to the generative capacities of spontaneous mutagenesis; and the correlation between genome organization and evolvability. We show that adaptive evolution is feasible only when the mutation rate is fine-tuned to a specific range of values and the structures of the genome and genes are optimized in a certain way. Our quantitative analysis has demonstrated that the rate of evolution of novelty depends on several parameters, such as genome size, the length of a module, the size of the adjacent nonfunctional DNA spacers, and the mutation rate at various genomic scales. We evaluated the efficiency of some mechanisms that increase evolvability: bias in the spectrum of mutation rates towards small mutations, and the availability and size of nonfunctional DNA spacers. We show that the probability of successful duplication and insertion of a copy of a functional module increases by several orders of magnitude depending on the length of the spacers flanking the module. We infer that the adaptive evolution of multicellular organisms has become feasible because of the abundance of nonfunctional DNA spacers, particularly introns, in the genome. We also discuss possible reasons underlying evolutionary retention of the mechanisms that increase evolvability.     

A 24-residue presequence localizes human factor B to mitochondria

We reported previously that the human factor B precursor is a 215-amino acid polypeptide, the first 40 amino acid residues of which function as a mitochondrial targeting presequence [G.I. Belogrudov, Y. Hatefi, J. Biol. Chem. 277 (2002) 6097-6103]. Confocal microscopy of live HEK293 cells, transiently transfected with factor B constructs tagged at the C-terminus with green fluorescent protein (GFP) revealed that either a 40- or 25-residue presequence localized factor B to mitochondria. Indirect immunofluorescent labeling of fixed, permeabilized HEK293 cells that were transiently transfected with a construct lacking a presequence, showed diffuse, intracellular staining that was consistent with targeting of ectopically expressed factor B to cellular compartments distinct from the mitochondria. Mutants in which either Met(-25) or both Met(-25)/Met(-24) residues of the presequence were deleted exhibited decreased or undetectable levels, respectively, of the GFP-tagged factor B. The factor B presequence alone was shown to target a reporter polypeptide GFP to mitochondria. Our studies, therefore, demonstrate that a 24-residue presequence is sufficient to localize factor B to mitochondria, and suggest that the human factor B precursor is a 199-amino acid polypeptide.     

Targeting base excision repair to improve cancer therapies

Most commonly used cancer therapies, particularly ionizing radiation and certain classes of cytotoxic chemotherapies, cause cell death by damaging DNA. Base excision repair (BER) is the major system responsible for the removal of corrupt DNA bases and repair of DNA single strand breaks generated spontaneously and induced by exogenous DNA damaging factors such as certain cancer therapies. In this review, the physico-chemical properties of the proteins involved in BER are discussed with particular emphasis on molecular mechanisms coordinating repair processes. The aim of this review is to apply extensive knowledge that currently exists regarding the biochemical mechanisms involved in human BER to the molecular biology of current therapies for cancer. It is anticipated that the application of this knowledge will translate into the development of novel effective therapies for improving existing treatments such as radiation therapy and oxaliplatin chemotherapy.     

DNA polymerase delta-dependent repair of DNA single strand breaks containing 3'-end proximal lesions

Base excision repair (BER) is the major pathway for the repair of simple, non-bulky lesions in DNA that is initiated by a damage-specific DNA glycosylase. Several human DNA glycosylases exist that efficiently excise numerous types of lesions, although the close proximity of a single strand break (SSB) to a DNA adduct can have a profound effect on both BER and SSB repair. We recently reported that DNA lesions located as a second nucleotide 5′-upstream to a DNA SSB are resistant to DNA glycosylase activity and this study further examines the processing of these ‘complex’ lesions. We first demonstrated that the damaged base should be excised before SSB repair can occur, since it impaired processing of the SSB by the BER enzymes, DNA ligase IIIα and DNA polymerase β. Using human whole cell extracts, we next isolated the major activity against DNA lesions located as a second nucleotide 5′-upstream to a DNA SSB and identified it as DNA polymerase δ (Pol δ). Using recombinant protein we confirmed that the 3′-5′-exonuclease activity of Pol δ can efficiently remove these DNA lesions. Furthermore, we demonstrated that mouse embryonic fibroblasts, deficient in the exonuclease activity of Pol δ are partially deficient in the repair of these ‘complex’ lesions, demonstrating the importance of Pol δ during the repair of DNA lesions in close proximity to a DNA SSB, typical of those induced by ionizing radiation.