The Experts below are selected from a list of 297 Experts worldwide ranked by ideXlab platform
Myron F Goodman - One of the best experts on this subject based on the ideXlab platform.
-
a RecA protein surface required for activation of dna polymerase v
PLOS Genetics, 2015Co-Authors: Angela J Gruber, Kiyonobu Karata, Roger Woodgate, Aysen L Erdem, Grzegorz Sabat, Malgorzata Jaszczur, Dan D Vo, Tayla M Olsen, Myron F GoodmanAbstract:DNA polymerase V (pol V) of Escherichia coli is a translesion DNA polymerase responsible for most of the mutagenesis observed during the SOS response. Pol V is activated by transfer of a RecA subunit from the 3'-proximal end of a RecA nucleoprotein filament to form a functional complex called DNA polymerase V Mutasome (pol V Mut). We identify a RecA surface, defined by residues 112-117, that either directly interacts with or is in very close proximity to amino acid residues on two distinct surfaces of the UmuC subunit of pol V. One of these surfaces is uniquely prominent in the active pol V Mut. Several conformational states are populated in the inactive and active complexes of RecA with pol V. The RecA D112R and RecA D112R N113R double mutant proteins exhibit successively reduced capacity for pol V activation. The double mutant RecA is specifically defective in the ATP binding step of the activation pathway. Unlike the classic non-mutable RecA S117F (RecA1730), the RecA D112R N113R variant exhibits no defect in filament formation on DNA and promotes all other RecA activities efficiently. An important pol V activation surface of RecA protein is thus centered in a region encompassing amino acid residues 112, 113, and 117, a surface exposed at the 3'-proximal end of a RecA filament. The same RecA surface is not utilized in the RecA activation of the homologous and highly mutagenic RumA'2B polymerase encoded by the integrating-conjugative element (ICE) R391, indicating a lack of structural conservation between the two systems. The RecA D112R N113R protein represents a new separation of function mutant, proficient in all RecA functions except SOS mutagenesis.
-
the active form of dna polymerase v is umud 2 c RecA atp
Nature, 2009Co-Authors: Qingfei Jiang, Kiyonobu Karata, Roger Woodgate, Myron F GoodmanAbstract:DNA-damage-induced SOS mutations arise when Escherichia coli DNA polymerase (pol) V, activated by a RecA nucleoprotein filament (RecA*), catalyses translesion DNA synthesis. Here we address two longstanding enigmatic aspects of SOS mutagenesis, the molecular composition of mutagenically active pol V and the role of RecA*. We show that RecA* transfers a single RecA–ATP stoichiometrically from its DNA 3′-end to free pol V (UmuD′2C) to form an active mutasome (pol V Mut) with the composition UmuD′2C–RecA–ATP. Pol V Mut catalyses TLS in the absence of RecA* and deactivates rapidly upon dissociation from DNA. Deactivation occurs more slowly in the absence of DNA synthesis, while retaining RecA–ATP in the complex. Reactivation of pol V Mut is triggered by replacement of RecA–ATP from RecA*. Thus, the principal role of RecA* in SOS mutagenesis is to transfer RecA–ATP to pol V, and thus generate active mutasomal complex for translesion synthesis. Although it has been known that Escherichia coli DNA polymerase V is involved in the mutagenic process of limited DNA synthesis across a DNA lesion, the nature of the active complex and the requirement for an active filament of the strand exchange protein RecA have remained unclear. In this work, Goodman and colleagues have defined biochemically that active pol V consists of UmuD′2C, a single RecA monomer, and ATP. This ATP-bound RecA monomer cannot join UmuD′2C from solution but must be transferred from the RecA filament, thereby explaining its role in the process. Escherichia coli DNA polymerase (pol) V is involved in the mutagenic process of limited DNA synthesis across a DNA lesion, but the molecular composition of mutagenically active pol V and the importance of the RecA nucleoprotein filament RecA* have remained unclear. The biochemical role of RecA* is now defined.
-
RecA acts in trans to allow replication of damaged dna by dna polymerase v
Nature, 2006Co-Authors: Katharina Schlacher, Roger Woodgate, Myron F GoodmanAbstract:The DNA polymerase V (pol V) and RecA proteins are essential components of a mutagenic translesion synthesis pathway in Escherichia coli designed to cope with DNA damage. Previously, it has been assumed that RecA binds to the DNA template strand being copied. Here we show, however, that pol-V-catalysed translesion synthesis, in the presence or absence of the β-processivity-clamp, occurs only when RecA nucleoprotein filaments assemble or RecA protomers bind on separate single-stranded (ss)DNA molecules in trans. A 3′-proximal RecA filament end on trans DNA is essential for stimulation; however, synthesis is strengthened by further pol V–RecA interactions occurring elsewhere along a trans nucleoprotein filament. We suggest that trans-stimulation of pol V by RecA bound to ssDNA reflects a distinctive regulatory mechanism of mutation that resolves the paradox of RecA filaments assembled in cis on a damaged template strand obstructing translesion DNA synthesis despite the absolute requirement of RecA for SOS mutagenesis. A new model for the repair of damaged DNA in Escherichia coli could resolve earlier contradictory observations. Unrepaired DNA lesions can block genome replication, so the cell has defences to deal with such lesions when they are encountered by the replication machinery. In E. coli this involves pol V, a specialized DNA polymerase and RecA, a DNA repair protein. Previous work suggested that RecA binds to single-stranded DNA (ssDNA) on the same strand as the lesion, stimulating pol V to synthesize DNA across the damaged template. Now Schlacher et al. show that in fact RecA binds to a different ssDNA, and activates pol V 'in trans', not on the strand being copied.
Edward H. Egelman - One of the best experts on this subject based on the ideXlab platform.
-
Complexes of RecA with LexA and RecX differentiate between active and inactive RecA nucleoprotein filaments.
Journal of Molecular Biology, 2003Co-Authors: Margaret S VanLoock, Shixin Yang, Xiong Yu, Vitold E. Galkin, Hao Huang, S.s. Rajan, Wayne F. Anderson, Elizabeth A. Stohl, H. Steven Seifert, Edward H. EgelmanAbstract:The bacterial RecA protein has been the dominant model system for understanding homologous genetic recombination. Although a crystal structure of RecA was solved ten years ago, we still do not have a detailed understanding of how the helical filament formed by RecA on DNA catalyzes the recognition of homology and the exchange of strands between two DNA molecules. Recent structural and spectroscopic studies have suggested that subunits in the helical filament formed in the RecA crystal are rotated when compared to the active RecA-ATP-DNA filament. We examine RecA-DNA-ATP filaments complexed with LexA and RecX to shed more light on the active RecA filament. The LexA repressor and RecX, an inhibitor of RecA, both bind within the deep helical groove of the RecA filament. Residues on RecA that interact with LexA cannot be explained by the crystal filament, but can be properly positioned in an existing model for the active filament. We show that the strand exchange activity of RecA, which can be inhibited when RecX is present at very low stoichiometry, is due to RecX forming a block across the deep helical groove of the RecA filament, where strand exchange occurs. It has previously been shown that changes in the nucleotide bound to RecA are associated with large motions of RecA's C-terminal domain. Since RecX binds from the C-terminal domain of one subunit to the nucleotide-binding core of another subunit, a stabilization of RecA's C-terminal domain by RecX can likely explain the inhibition of RecA's ATPase activity by RecX.
-
domain structure and dynamics in the helical filaments formed by RecA and rad51 on dna
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Xiong Yu, S Jacobs, Stephen C West, Tomoko Ogawa, Edward H. EgelmanAbstract:Both the bacterial RecA protein and the eukaryotic Rad51 protein form helical nucleoprotein filaments on DNA that catalyze strand transfer between two homologous DNA molecules. However, only the ATP-binding cores of these proteins have been conserved, and this same core is also found within helicases and the F1-ATPase. The C-terminal domain of the RecA protein forms lobes within the helical RecA filament. However, the Rad51 proteins do not have the C-terminal domain found in RecA, but have an N-terminal extension that is absent in the RecA protein. Both the RecA C-terminal domain and the Rad51 N-terminal domain bind DNA. We have used electron microscopy to show that the lobes of the yeast and human Rad51 filaments appear to be formed by N-terminal domains. These lobes are conformationally flexible in both RecA and Rad51. Within RecA filaments, the change between the “active” and “inactive” states appears to mainly involve a large movement of the C-terminal lobe. The N-terminal domain of Rad51 and the C-terminal domain of RecA may have arisen from convergent evolution to play similar roles in the filaments.
-
Comparison of bacteriophage T4 UvsX and human Rad51 filaments suggests that RecA-like polymers may have evolved independently11Edited by M. Belfort
Journal of Molecular Biology, 2001Co-Authors: Shixin Yang, Margaret S VanLoock, Xiong Yu, Edward H. EgelmanAbstract:The UvsX protein from bacteriophage T4 is a member of the RecA/Rad51/RadA family of recombinases active in homologous genetic recombination. Like RecA, Rad51 and RadA, UvsX forms helical filaments on DNA. We have used electron microscopy and a novel method for image analysis of helical filaments to show that UvsX-DNA filaments exist in two different conformations: an ADP state and an ATP state. As with RecA protein, these two states have a large difference in pitch. Remarkably, even though UvsX is only weakly homologous to RecA, both UvsX filament states are more similar to the RecA crystal structure than are RecA-DNA filaments. We use this similarity to fit the RecA crystal structure into the UvsX filament, and show that two of the three previously described blocks of similarity between UvsX and RecA are involved in the subunit-subunit interface in both the UvsX filament and the RecA crystal filament. Conversely, we show that human Rad51-DNA filaments have a different subunit-subunit interface than is present in the RecA crystal, and this interface involves two blocks of sequence similarity between Rad51 and RecA that do not overlap with those found between UvsX and RecA. This suggests that helical filaments in the RecA/Rad51/RadA family may have arisen from convergent evolution, with a conserved core structure that has assembled into multimeric filaments in a number of different ways.
-
Removal of the RecA C-terminus results in a conformational change in the RecA-DNA filament
Journal of Structural Biology, 1991Co-Authors: Xiong Yu, Edward H. EgelmanAbstract:Abstract The Escherichia coli RecA protein catalyzes homologous recombination of DNA molecules, and the active form of the protein is a helical polymer that it forms around DNA. Previous image analysis of electron micrographs has revealed the RecA protein to be organized into two domains or lobes within the RecA-DNA filament. We have now been able to show that a small modification of the RecA protein by proteolysis results in a significant shift in the internal mass in the RecA filament. We have cleaved approximately 18 residues from the C-terminus of the RecA protein, producing a roughly 36K MW RecA core protein that binds DNA and polymerizes normally. A three-dimensional reconstruction of this complex has been computed, and has been compared with a previous reconstruction of the intact protein. The main difference is consistent with a 15 A outward movement of the lobe that was at an inner radius in the wild-type protein. These observations yield additional evidence about the conformational flexibility of the RecA filament, and will aid in understanding the structural mechanics and dynamics of the RecA filament.
K. Muniyappa - One of the best experts on this subject based on the ideXlab platform.
-
The Anionic Phospholipids in the Plasma Membrane Play an Important Role in Regulating the Biochemical Properties and Biological Functions of RecA Proteins
Biochemistry, 2019Co-Authors: Deepika Prasad, K. MuniyappaAbstract:Escherichia coli RecA (EcRecA) forms discrete foci that cluster at cell poles during normal growth, which are redistributed along the filamented cell axis upon induction of the SOS response. The plasma membrane is thought to act as a scaffold for EcRecA foci, thereby playing an important role in RecA-dependent homologous recombination. In addition, in vivo and in vitro studies demonstrate that EcRecA binds strongly to the anionic phospholipids. However, there have been almost no data on the association of mycobacterial RecA proteins with the plasma membrane and the effects of membrane components on their function. Here, we show that mycobacterial RecA proteins specifically interact with phosphatidylinositol and cardiolipin among other anionic phospholipids; however, they had no effect on the ability of RecA proteins to bind single-stranded DNA. Interestingly, phosphatidylinositol and cardiolipin impede the DNA-dependent ATPase activity of RecA proteins, although ATP binding is not affected. Furthermore, t...
-
suramin is a potent and selective inhibitor of mycobacterium tuberculosis RecA protein and the sos response RecA as a potential target for antibacterial drug discovery
Journal of Antimicrobial Chemotherapy, 2014Co-Authors: Astha Nautiyal, Neelakanteshwar K Patil, K. MuniyappaAbstract:In eubacteria, RecA is essential for recombinational DNA repair and for stalled replication forks to resume DNA synthesis. Recent work has implicated a role for RecA in the development of antibiotic resistance in pathogenic bacteria. Consequently, our goal is to identify and characterize small-molecule inhibitors that target RecA both in vitro and in vivo. We employed ATPase, DNA strand exchange and LexA cleavage assays to elucidate the inhibitory effects of suramin on Mycobacterium tuberculosis RecA. To gain insights into the mechanism of suramin action, we directly visualized the structure of RecA nucleoprotein filaments by atomic force microscopy. To determine the specificity of suramin action in vivo, we investigated its effect on the SOS response by pull-down and western blot assays as well as for its antibacterial activity. We show that suramin is a potent inhibitor of DNA strand exchange and ATPase activities of bacterial RecA proteins with IC50 values in the low micromolar range. Additional evidence shows that suramin inhibits RecA-catalysed proteolytic cleavage of the LexA repressor. The mechanism underlying such inhibitory actions of suramin involves its ability to disassemble RecA-single-stranded DNA filaments. Notably, suramin abolished ciprofloxacin-induced RecA gene expression and the SOS response and augmented the bactericidal action of ciprofloxacin. Our findings suggest a strategy to chemically disrupt the vital processes controlled by RecA and hence the promise of small molecules for use against drug-susceptible as well as drug-resistant strains of M. tuberculosis for better infection control and the development of new therapies.
-
Mycobacterium smegmatis RecA protein is structurally similar to but functionally distinct from Mycobacterium tuberculosis RecA
Proteins, 2003Co-Authors: N. Ganesh, K. MuniyappaAbstract:In eubacteria, RecA proteins belong to a large superfamily of evolutionarily conserved, filament-forming, functional homologs of DNA strand exchange proteins. Here, we report the functional characterization of Mycobacterium smegmatis (Ms) and Mycobacterium tuberculosis (Mt) RecA proteins. Although in some respects Ms and Mt RecA proteins are structural and functional homologs of Escherichia coli (Ec) RecA, there are significant differences as well. The single-stranded DNA-binding property of RecA proteins was analyzed by electrophoretic mobility shift assays. We observed that Ms or Mt RecA proteins bound single-stranded DNA in a manner distinct from that of Ec RecA: The former two were able to form protein-DNA complexes in the presence of high salt. Further experiments indicated that Ms or Mt RecA proteins catalyzed adenosine triphosphate hydrolysis at approximately comparable rates across a wide range of pHs. Significantly, DNA strand invasion promoted by Ms or Mt RecA proteins displayed similar kinetics but distinctly different pH profiles. In contrast to MtRecA, MsRecA by itself was unable to form joint molecules across a wide range of pHs. However, regardless of the order in which SSB was added, it was able to stimulate MsRecA to form joint molecules within a narrow pH range, indicating that SSB is a required accessory factor. Together, these results provide a source of sharp contrast between EcRecA and mycobacterial RecAs on the one hand and Mt and Ms RecA proteins on the other.
James L Keck - One of the best experts on this subject based on the ideXlab platform.
-
structural and functional studies of h seropedicae RecA protein insights into the polymerization of RecA protein as nucleoprotein filament
PLOS ONE, 2016Co-Authors: Wellington Claiton Leite, Carolina W Galvao, Sergio Da Costa Saab, J Iulek, Rafael Mazer Etto, Maria B R Steffens, Sindhu Chittenipattu, Tyler Stanage, James L KeckAbstract:The bacterial RecA protein plays a role in the complex system of DNA damage repair. Here, we report the functional and structural characterization of the Herbaspirillum seropedicae RecA protein (HsRecA). HsRecA protein is more efficient at displacing SSB protein from ssDNA than Escherichia coli RecA protein. HsRecA also promotes DNA strand exchange more efficiently. The three dimensional structure of HsRecA-ADP/ATP complex has been solved to 1.7 A resolution. HsRecA protein contains a small N-terminal domain, a central core ATPase domain and a large C-terminal domain, that are similar to homologous bacterial RecA proteins. Comparative structural analysis showed that the N-terminal polymerization motif of archaeal and eukaryotic RecA family proteins are also present in bacterial RecAs. Reconstruction of electrostatic potential from the hexameric structure of HsRecA-ADP/ATP revealed a high positive charge along the inner side, where ssDNA is bound inside the filament. The properties of this surface may explain the greater capacity of HsRecA protein to bind ssDNA, forming a contiguous nucleoprotein filament, displace SSB and promote DNA exchange relative to EcRecA. Our functional and structural analyses provide insight into the molecular mechanisms of polymerization of bacterial RecA as a helical nucleoprotein filament.
-
Structural and Functional Studies of H. seropedicae RecA Protein – Insights into the Polymerization of RecA Protein as Nucleoprotein Filament
PLOS ONE, 2016Co-Authors: Wellington Claiton Leite, Carolina W Galvao, Sergio Da Costa Saab, J Iulek, Rafael Mazer Etto, Maria B R Steffens, Tyler Stanage, Sindhu Chitteni-pattu, James L KeckAbstract:The bacterial RecA protein plays a role in the complex system of DNA damage repair. Here, we report the functional and structural characterization of the Herbaspirillum seropedicae RecA protein (HsRecA). HsRecA protein is more efficient at displacing SSB protein from ssDNA than Escherichia coli RecA protein. HsRecA also promotes DNA strand exchange more efficiently. The three dimensional structure of HsRecA-ADP/ATP complex has been solved to 1.7 A resolution. HsRecA protein contains a small N-terminal domain, a central core ATPase domain and a large C-terminal domain, that are similar to homologous bacterial RecA proteins. Comparative structural analysis showed that the N-terminal polymerization motif of archaeal and eukaryotic RecA family proteins are also present in bacterial RecAs. Reconstruction of electrostatic potential from the hexameric structure of HsRecA-ADP/ATP revealed a high positive charge along the inner side, where ssDNA is bound inside the filament. The properties of this surface may explain the greater capacity of HsRecA protein to bind ssDNA, forming a contiguous nucleoprotein filament, displace SSB and promote DNA exchange relative to EcRecA. Our functional and structural analyses provide insight into the molecular mechanisms of polymerization of bacterial RecA as a helical nucleoprotein filament.
Roger Woodgate - One of the best experts on this subject based on the ideXlab platform.
-
a RecA protein surface required for activation of dna polymerase v
PLOS Genetics, 2015Co-Authors: Angela J Gruber, Kiyonobu Karata, Roger Woodgate, Aysen L Erdem, Grzegorz Sabat, Malgorzata Jaszczur, Dan D Vo, Tayla M Olsen, Myron F GoodmanAbstract:DNA polymerase V (pol V) of Escherichia coli is a translesion DNA polymerase responsible for most of the mutagenesis observed during the SOS response. Pol V is activated by transfer of a RecA subunit from the 3'-proximal end of a RecA nucleoprotein filament to form a functional complex called DNA polymerase V Mutasome (pol V Mut). We identify a RecA surface, defined by residues 112-117, that either directly interacts with or is in very close proximity to amino acid residues on two distinct surfaces of the UmuC subunit of pol V. One of these surfaces is uniquely prominent in the active pol V Mut. Several conformational states are populated in the inactive and active complexes of RecA with pol V. The RecA D112R and RecA D112R N113R double mutant proteins exhibit successively reduced capacity for pol V activation. The double mutant RecA is specifically defective in the ATP binding step of the activation pathway. Unlike the classic non-mutable RecA S117F (RecA1730), the RecA D112R N113R variant exhibits no defect in filament formation on DNA and promotes all other RecA activities efficiently. An important pol V activation surface of RecA protein is thus centered in a region encompassing amino acid residues 112, 113, and 117, a surface exposed at the 3'-proximal end of a RecA filament. The same RecA surface is not utilized in the RecA activation of the homologous and highly mutagenic RumA'2B polymerase encoded by the integrating-conjugative element (ICE) R391, indicating a lack of structural conservation between the two systems. The RecA D112R N113R protein represents a new separation of function mutant, proficient in all RecA functions except SOS mutagenesis.
-
the active form of dna polymerase v is umud 2 c RecA atp
Nature, 2009Co-Authors: Qingfei Jiang, Kiyonobu Karata, Roger Woodgate, Myron F GoodmanAbstract:DNA-damage-induced SOS mutations arise when Escherichia coli DNA polymerase (pol) V, activated by a RecA nucleoprotein filament (RecA*), catalyses translesion DNA synthesis. Here we address two longstanding enigmatic aspects of SOS mutagenesis, the molecular composition of mutagenically active pol V and the role of RecA*. We show that RecA* transfers a single RecA–ATP stoichiometrically from its DNA 3′-end to free pol V (UmuD′2C) to form an active mutasome (pol V Mut) with the composition UmuD′2C–RecA–ATP. Pol V Mut catalyses TLS in the absence of RecA* and deactivates rapidly upon dissociation from DNA. Deactivation occurs more slowly in the absence of DNA synthesis, while retaining RecA–ATP in the complex. Reactivation of pol V Mut is triggered by replacement of RecA–ATP from RecA*. Thus, the principal role of RecA* in SOS mutagenesis is to transfer RecA–ATP to pol V, and thus generate active mutasomal complex for translesion synthesis. Although it has been known that Escherichia coli DNA polymerase V is involved in the mutagenic process of limited DNA synthesis across a DNA lesion, the nature of the active complex and the requirement for an active filament of the strand exchange protein RecA have remained unclear. In this work, Goodman and colleagues have defined biochemically that active pol V consists of UmuD′2C, a single RecA monomer, and ATP. This ATP-bound RecA monomer cannot join UmuD′2C from solution but must be transferred from the RecA filament, thereby explaining its role in the process. Escherichia coli DNA polymerase (pol) V is involved in the mutagenic process of limited DNA synthesis across a DNA lesion, but the molecular composition of mutagenically active pol V and the importance of the RecA nucleoprotein filament RecA* have remained unclear. The biochemical role of RecA* is now defined.
-
RecA acts in trans to allow replication of damaged dna by dna polymerase v
Nature, 2006Co-Authors: Katharina Schlacher, Roger Woodgate, Myron F GoodmanAbstract:The DNA polymerase V (pol V) and RecA proteins are essential components of a mutagenic translesion synthesis pathway in Escherichia coli designed to cope with DNA damage. Previously, it has been assumed that RecA binds to the DNA template strand being copied. Here we show, however, that pol-V-catalysed translesion synthesis, in the presence or absence of the β-processivity-clamp, occurs only when RecA nucleoprotein filaments assemble or RecA protomers bind on separate single-stranded (ss)DNA molecules in trans. A 3′-proximal RecA filament end on trans DNA is essential for stimulation; however, synthesis is strengthened by further pol V–RecA interactions occurring elsewhere along a trans nucleoprotein filament. We suggest that trans-stimulation of pol V by RecA bound to ssDNA reflects a distinctive regulatory mechanism of mutation that resolves the paradox of RecA filaments assembled in cis on a damaged template strand obstructing translesion DNA synthesis despite the absolute requirement of RecA for SOS mutagenesis. A new model for the repair of damaged DNA in Escherichia coli could resolve earlier contradictory observations. Unrepaired DNA lesions can block genome replication, so the cell has defences to deal with such lesions when they are encountered by the replication machinery. In E. coli this involves pol V, a specialized DNA polymerase and RecA, a DNA repair protein. Previous work suggested that RecA binds to single-stranded DNA (ssDNA) on the same strand as the lesion, stimulating pol V to synthesize DNA across the damaged template. Now Schlacher et al. show that in fact RecA binds to a different ssDNA, and activates pol V 'in trans', not on the strand being copied.
