A mutation in E. coli SSB protein (W54S) alters intra-tetramer negative cooperativity and inter-tetramer positive cooperativity for single-stranded DNA binding

Ferrari, Marilyn E.; Fang, Junguo; Lohman, Timothy M.

doi:10.1016/s0301-4622(96)02223-5

Cited by 23 publications

(18 citation statements)

References 40 publications

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“…In Escherichia coli , the SSB protein can exist in two states, (SSB) 35 and (SSB) 65 , that bind ssDNA with different degrees of cooperativity (58). Studies have revealed that the high positive cooperativity state (SSB) 35 is stabilized by the W54S mutation (59,60). Interestingly, when the gene coding for this mutated protein is expressed in E. coli , cells exhibit slow growth and increased UV sensitivity (59).…”

Section: Discussionmentioning

confidence: 99%

A conserved lysine residue of plant Whirly proteins is necessary for higher order protein assembly and protection against DNA damage

Cappadocia

Parent

Zampini

et al. 2011

Nucleic Acids Research

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All organisms have evolved specialized DNA repair mechanisms in order to protect their genome against detrimental lesions such as DNA double-strand breaks. In plant organelles, these damages are repaired either through recombination or through a microhomology-mediated break-induced replication pathway. Whirly proteins are modulators of this second pathway in both chloroplasts and mitochondria. In this precise pathway, tetrameric Whirly proteins are believed to bind single-stranded DNA and prevent spurious annealing of resected DNA molecules with other regions in the genome. In this study, we add a new layer of complexity to this model by showing through atomic force microscopy that tetramers of the potato Whirly protein WHY2 further assemble into hexamers of tetramers, or 24-mers, upon binding long DNA molecules. This process depends on tetramer–tetramer interactions mediated by K67, a highly conserved residue among plant Whirly proteins. Mutation of this residue abolishes the formation of 24-mers without affecting the protein structure or the binding to short DNA molecules. Importantly, we show that an Arabidopsis Whirly protein mutated for this lysine is unable to rescue the sensitivity of a Whirly-less mutant plant to a DNA double-strand break inducing agent.

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Section: Discussionmentioning

confidence: 99%

A conserved lysine residue of plant Whirly proteins is necessary for higher order protein assembly and protection against DNA damage

Cappadocia

Parent

Zampini

et al. 2011

Nucleic Acids Research

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“…6E). We used a variant SSB, SSB W54S, which binds dT 35 with one-five hundredth the avidity of the wild type (39), to determine whether the ssDNA binding activity of SSB was required for this inhibition. This proved to be the case (Fig.…”

Section: Reca Has Little Effect On Stalled Forksmentioning

confidence: 99%

Regression of Replication Forks Stalled by Leading-strand Template Damage

Gupta

Yeeles

Marians

2014

Journal of Biological Chemistry

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“…higher oligomers when bound to the nucleic acid, and it has been proposed that this includes the formation of octamers through the dimerization of tetramers (48); similar octameric forms were also suggested for the vaccinia virus I3L gene product (49). However, generally little sequence similarity of EcoSSB and related proteins with the single-stranded DNAbinding proteins of animal viruses are found (50), and it is not clear if NSP2 and EcoSSB share more structural motifs than a high fraction of ␤-sheet.…”

Section: Fig 7 CD Spectra Of Nsp2mentioning

confidence: 99%

Rotavirus Nonstructural Protein NSP2 Self-assembles into Octamers That Undergo Ligand-induced Conformational Changes

Schuck

Taraporewala

McPhie³

et al. 2001

Journal of Biological Chemistry

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The nonstructural protein NSP2 is a component of the rotavirus replication machinery and binds singlestranded RNA cooperatively, with high affinity, and independent of sequence. Recently, NSP2 has been shown to form multimers and to possess an NTPase activity, but its precise function remains unclear. In the present study, we have characterized the solution structure of recombinant NSP2 by velocity and equilibrium ultracentrifugation, dynamic light scattering, and circular dichroism spectroscopy. We found that NSP2 exists as an octamer, which is functional in the binding of RNA and ADP. In the presence of magnesium, a partial dissociation of the octamer into smaller oligomers was observed. This was reversed by binding of ADP and RNA. We observed an increased sedimentation rate in the presence of ADP and a nonhydrolyzable ATP analogue, which suggests a change toward a significantly more compact octameric conformation. The secondary structure of NSP2 showed a high fraction of ␤-sheet, Rotavirus is a significant cause of disease in humans and animals. It is a member of the Reoviridae, and its genome consists of 11 segments of double-stranded RNA, which codes for six structural and six nonstructural proteins. The structural proteins include VP2, VP6, and VP7, which form the triple layered icosahedral virion capsid, the spike protein VP4, the RNA polymerase VP1, and the multifunctional capping enzyme VP3 (1) (for a review, see Ref.2). Many studies have addressed the properties of the structural proteins, for example their spatial configuration in the virus particle using cryo-EM (3, 4), their antigenicity, and their role in viral entry, replication, or morphogenesis. Unfortunately, much less is known about the nonstructural proteins that are expressed and left behind in the infected cells.Although it has been shown that these nonstructural proteins are not essential for replicase activity in vitro (5), they are important in several aspects of the replication cycle of the virus in vivo. Some of the more intensively studied nonstructural proteins include, for example, NSP4, which has a membranedestabilizing activity and assists in the budding of newly synthesized inner capsid particles into the lumen of the endoplasmic reticulum (6, 7), where they acquire the outer coat protein VP7. NSP3 binds to the 3Ј-end of the viral mRNA and interacts with eukaryotic translation initiation factor eIF4G to enhance efficiency of translation (8). NSP2 and NSP5 interact to form viroplasms, large inclusions in the cytoplasm where core-like replication intermediates (core RIs) 1 are assembled and RNA replication takes place (2, 9, 10). As was shown in a study of a temperature-sensitive mutant, NSP2 is required for the formation of the viroplasm and is also essential in the synthesis of double-stranded RNA in vivo (11). However, little is known about the mechanism underlying this observation and the function of NSP2 on a molecular level.NSP2 is a 35-kDa protein that forms homomultimers and interacts with the RNA polymerase VP1 (12, 17). F...

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A mutation in E. coli SSB protein (W54S) alters intra-tetramer negative cooperativity and inter-tetramer positive cooperativity for single-stranded DNA binding

Cited by 23 publications

References 40 publications

A conserved lysine residue of plant Whirly proteins is necessary for higher order protein assembly and protection against DNA damage

A conserved lysine residue of plant Whirly proteins is necessary for higher order protein assembly and protection against DNA damage

Regression of Replication Forks Stalled by Leading-strand Template Damage

Rotavirus Nonstructural Protein NSP2 Self-assembles into Octamers That Undergo Ligand-induced Conformational Changes

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