Intersubunit Proximity of Residues in the RecA Protein As Shown by Engineered Disulfide Cross-Links

Skiba, Mark C.; Logan, Karen M.; Knight, Kendall L.

doi:10.1021/bi991118z

Cited by 18 publications

(10 citation statements)

References 25 publications

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“…From the yeast proteasome structure (PDB entry 1RYP), we identified Pre9 residues that contacted Pre8 and, by sequence alignment, were conserved in Pre6. The β carbons of Pre8‐Lys160 and Pre9‐Leu56 are within 4.9 Å of each other, close to β‐carbon separations seen in natural disulfide bonds (Skiba et al , 1999), so these residues were changed to cysteines.…”

Section: Resultsmentioning

confidence: 62%

Plasticity in eucaryotic 20S proteasome ring assembly revealed by a subunit deletion in yeast

Velichutina¹,

Connerly²,

Arendt³

et al. 2004

EMBO J

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The 20S proteasome is made up of four stacked heptameric rings, which in eucaryotes assemble from 14 different but related subunits. The rules governing subunit assembly and placement are not understood. We show that a different kind of proteasome forms in yeast when the Pre9/a3 subunit is deleted. Purified pre9D proteasomes show a two-fold enrichment for the Pre6/a4 subunit, consistent with the presence of an extra copy of Pre6 in each outer ring. Based on disulfide engineering and structure-guided suppressor analyses, Pre6 takes the position normally occupied by Pre9, a substitution that depends on a network of intersubunit salt bridges. When Arabidopsis PAD1/a4 is expressed in yeast, it complements not only pre6D but also pre6D pre9D mutants; therefore, the plant a4 subunit also can occupy multiple positions in a functional yeast proteasome. Importantly, biogenesis of proteasomes is delayed at an early stage in pre9D cells, suggesting an advantage for Pre9 over Pre6 incorporation at the a3 position that facilitates correct assembly.

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Section: Resultsmentioning

confidence: 62%

Plasticity in eucaryotic 20S proteasome ring assembly revealed by a subunit deletion in yeast

Velichutina¹,

Connerly²,

Arendt³

et al. 2004

EMBO J

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“…Many studies have shown that the oligomeric state of free RecA protein is exquisitely sensitive to a variety of solution conditions including protein concentration, ionic strength, and temperature and that at the protein concentrations typically used in DNA strand exchange assays, RecA exists as a complex mix of various oligomeric forms (9)(10)(11)(12)(27)(28)(29)(30). The fact that the RecA-FD protein functions both in ViVo and in Vitro demonstrates that dimeric RecA can serve as the nucleating unit for filament assembly onto ssDNA.…”

Section: Discussionmentioning

confidence: 99%

RecA Dimers Serve as a Functional Unit for Assembly of Active Nucleoprotein Filaments

et al. 2006

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All RecA-like recombinase enzymes catalyze DNA strand exchange as elongated filaments on DNA. Despite numerous biochemical and structural studies of RecA and the related Rad51 and RadA proteins, the unit oligomer(s) responsible for nucleoprotein filament assembly and coordinated filament activity remains undefined. We have created a RecA fused dimer protein and show that it maintains in vivo DNA repair and LexA co-protease activities, as well as in vitro ATPase and DNA strand exchange activities. Our results support the idea that dimeric RecA is an important functional unit both for assembly of nucleoprotein filaments as well as their coordinated activity during the catalysis of homologous recombination.The bacterial RecA protein is the founding member of a family of enzymes that catalyzes recombination between homologous DNA substrates, a family which includes the phage T4 UvsX, yeast Rad51, archael RadA and human Rad51 proteins (1,2). To perform this function these recombinase enzymes form a helical filament that assembles cooperatively on a singlestranded region of DNA resulting in the formation of an active nucleoprotein complex that subsequently interacts with a dsDNA substrate to catalyze a search for homology and DNA strand exchange (1,3,4). Early biochemical studies of RecA revealed that in the absence of substrate DNA the protein exists in solution as a complex heterogeneous mix of various oligomers (5-8) and later work showed that this mix included monomers, ring-shaped hexamers/heptamers, short filaments ranging in length from 0.03 to 0.15 μm as well as bundles of filaments (9-12).The RecA nucleoprotein filament assembles in an ATP-dependent, highly cooperative manner (13-16). Kinetic analysis of nucleoprotein filament assembly has shown that RecA polymerization onto ssDNA can be divided into two major steps, i. nucleation, which is rate limiting, and ii. filament extension (17). However, the oligomeric heterogeneity of RecA in these studies precluded identification of a specific RecA oligomer, e.g. monomers or dimers, as being an obligatory component of the assembly process, or whether various larger RecA oligomers, e.g. trimers or hexamers, could participate in filament assembly. It was also found that RecA oligomers formed in the absence of ATP or ssDNA are not directly interconvertible tel. -(508) EXPERIMENTAL PROCEDURES Expression ConstructsThe gene encoding wild type recA was expressed using a previously described construct, pTRecA420, in which recA is regulated by the tac promoter (20,21). The gene encoding the fused RecA dimer (recA-FD) was constructed as follows. Two point mutations, Lys6Ala and Arg28Ala, were introduced into the wild type recA gene carried in plasmid pTRecA420 using the QuikChange protocol (Stratagene). This mutant recA gene was PCR amplified using a top strand primer that overlapped the NcoI restriction site (underlined) at the fMet initiation codon (5′-GG AGT GAT GCC ATG GCT ATC GAC G -3′) and a bottom strand primer that adds a five-residue linker (Gly 3 Ser 2 )...

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“…Two highly conserved consecutive glycine residues (Karlin & Brocchieri, 1996), critical for RecA function (Hörtnagel et al 1999) and situated at the C-terminal junction between the loop and the protein core may participate in this flexibility (see also De Zutter et al 2001). Based on engineered disulfide cross-linking and mutation studies, residue Phe217 has been proposed to mediate a change in loop conformation via its insertion into a hydrophobic pocket, where it would bind more tightly than in its crystal position (Skiba et al 1999 ;De Zutter et al 2001). Phe217 belongs to the small helix G following loop L2 and close to the nucleotide-binding site (Fig.…”

Section: Transmission Of Structural Information Along the Filamentmentioning

confidence: 99%

Geometry of the DNA strands within the RecA nucleofilament: role in homologous recombination

Prévost

Takahashi

2003

Quart. Rev. Biophys.

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Homologous recombination consists of exchanging DNA strands of identical or almost identical sequence. This process is important for both DNA repair and DNA segregation. In prokaryotes, it involves the formation of long helical filaments of the RecA protein on DNA. These filaments incorporate double-stranded DNA from the cell's genetic material, recognize sequence homology and promote strand exchange between the two DNA segments. DNA processing by these nucleofilaments is characterized by large amplitude deformations of the double helix, which is stretched by 50% and unwound by 40% with respect to B-DNA. In this article, information concerning the structure and interactions of the RecA, DNA and ATP molecules involved in DNA strand exchange is gathered and analyzed to present a view of their possible arrangement within the filament, their behavior during strand exchange and during ATP hydrolysis, the mechanism of RecA-promoted DNA deformation and the role of DNA deformation in the process of homologous recombination. In particular, the unusual characteristics of DNA within the RecA filament are compared to the DNA deformations locally induced by architectural proteins which bind in the DNA minor groove. The possible role and location of two flexible loops of RecA are discussed.

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Intersubunit Proximity of Residues in the RecA Protein As Shown by Engineered Disulfide Cross-Links

Cited by 18 publications

References 25 publications

Plasticity in eucaryotic 20S proteasome ring assembly revealed by a subunit deletion in yeast

Plasticity in eucaryotic 20S proteasome ring assembly revealed by a subunit deletion in yeast

RecA Dimers Serve as a Functional Unit for Assembly of Active Nucleoprotein Filaments

Geometry of the DNA strands within the RecA nucleofilament: role in homologous recombination

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