Dynamics of Bacteriophage T4 Presynaptic Filament Assembly from Extrinsic Fluorescence Measurements of Gp32-Single-stranded DNA Interactions

Liu, Jie; Qian, Na; Morrical, Scott W.

doi:10.1074/jbc.m604349200

Cited by 29 publications

(84 citation statements)

References 54 publications

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“…It has been estimated that UvsY engages 3-4 nt per protomer (14,18,26), and the protomer-protomer spacing in the region is 21 Å, consistent with 3-4 linearly bound nucleotides. SPR analyses also support earlier observations (15,17,27) that ssDNA, gp32, and UvsY can form a ternary complex that mediates the gp32/UvsX exchange, and we constructed a model of this putative complex (Fig. 6A).…”

Section: Discussionsupporting

confidence: 61%

Structure and mechanism of the phage T4 recombination mediator protein UvsY

Gajewski

Waddell

Vaithiyalingam

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The UvsY recombination mediator protein is critical for efficient homologous recombination in bacteriophage T4 and is the functional analog of the eukaryotic Rad52 protein. During T4 homologous recombination, the UvsX recombinase has to compete with the prebound gp32 single-stranded binding protein for DNA-binding sites and UvsY stimulates this filament nucleation event. We report here the crystal structure of UvsY in four similar open-barrel heptameric assemblies and provide structural and biophysical insights into its function. The UvsY heptamer was confirmed in solution by centrifugation and light scattering, and thermodynamic analyses revealed that the UvsY-ssDNA interaction occurs within the assembly via two distinct binding modes. Using surface plasmon resonance, we also examined the binding of UvsY to both ssDNA and the ssDNAgp32 complex. These analyses confirmed that ssDNA can bind UvsY and gp32 independently and also as a ternary complex. They also showed that residues located on the rim of the heptamer are required for optimal binding to ssDNA, thus identifying the putative ssDNAbinding surface. We propose a model in which UvsY promotes a helical ssDNA conformation that disfavors the binding of gp32 and initiates the assembly of the ssDNA-UvsX filament.homologous recombination | structural modification | DNA binding | DNA architecture | crystallography H omologous recombination (HR) involves the exchange of strands between homologous DNA molecules and has fundamental roles in double-stranded DNA (dsDNA) break repair, the rescue of stalled replication forks, and recombination-dependent replication (1). Defects in HR are associated with genetic instability, chromosomal abnormalities, and cancer (1). HR is performed by an ATP-dependent recombinase, RecA in prokaryotes and Rad51 in eukaryotes, that creates a filament with approximately sixfold helical symmetry. The filament first binds single-stranded DNA (ssDNA) and then samples incoming dsDNA to search for homology and promote the exchange reaction (2, 3). Key insights into the mechanism have been provided by structural (4) and dynamics (5, 6) studies of the HR filament. ssDNA-binding proteins, RPA in eukaryotes and SSB in prokaryotes, protect the ssDNA from nucleases and remove secondary structures during HR, but they also block the binding of the recombinase (3). This obstacle is overcome by the recombination mediator proteins (RMPs) (7), Rad52 in eukaryotes and RecOR in prokaryotes, that stimulate the exchange of the ssDNA-binding proteins for the recombinase.Phage T4 is able to process DNA in isolation from the host Escherichia coli by encoding all of the necessary DNA metabolic proteins. The T4 UvsXYW system encodes the core HR machinery (8) comprising UvsX (the recombinase), UvsW (the SF2 remodeling helicase), and UvsY (the RMP). Together with the T4 ssDNA-binding protein gp32 (9, 10), these three proteins can efficiently perform HR in an in vitro reconstituted system (11). UvsY is a 15.8-kDa protein with properties that are consistent with its rol...

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Section: Discussionsupporting

confidence: 61%

Structure and mechanism of the phage T4 recombination mediator protein UvsY

Gajewski

Waddell

Vaithiyalingam

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…This observation might be explained if wildtype UvsX initiates or promotes the excess of templated mutagenesis in the 32mms mutant and this effect is blocked by wild-type gp32 but not by gp32mms. Interactions between gp32 and UvsX are well documented and can be either cooperative or competitive, depending on the parameter and experimental conditions (Villemain et al 2000;Liu et al 2006). For instance, when gp32 is defective, UvsX might promote the annealing of a melted primer terminus to the other parental strand, a mutagenic pathway, rather than to the other daughter strand, an antimutagenic pathway.…”

Section: Resultsmentioning

confidence: 99%

Templated Mutagenesis in Bacteriophage T4 Involving Imperfect Direct or Indirect Sequence Repeats

Schultz

Drake

2008

Genetics

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Some mutations arise in association with a potential sequence donor that consists of an imperfect direct or reverse repeat. Many such mutations are complex; that is, they consist of multiple close sequence changes. Current models posit that the primer terminus of a replicating DNA molecule dissociates, reanneals with an ectopic template, extends briefly, and then returns to the cognate template, bringing with it a locally different sequence; alternatively, a hairpin structure may form the mutational intermediate when processed by mismatch repair. This process resembles replication repair, in which primer extension is blocked by a lesion in the template; in this case, the ectopic template is the other daughter strand, and the result is errorfree bypass of the lesion. We previously showed that mutations that impair replication repair can enhance templated mutagenesis. We show here that the intensity of templated mutation can be exquisitely sensitive to its local sequence, that the donor and recipient arms of an imperfect inverse repeat can exchange roles, and that double mutants carrying two alleles, each affecting both templated mutagenesis and replication repair, can have unexpected phenotypes. We also record an instance in which the mutation rates at two particular sites change concordantly with a distant sequence change, but in a manner that appears unrelated to templated mutagenesis. T EMPLATED mutations are initiated when a DNA primer strand dissociates from its cognate template and anneals with a fragment of complementary sequence in an ectopic template. The relocated primer may be extended, acquiring a short sequence that is not fully complementary to the original template, and then reanneal with its cognate template. If the acquired noncomplementary bases escape correctly oriented proofreading and mismatch repair, mutations will result. Lynn Ripley (1982) described two models for templated mutagenesis based on imperfect palindromic repeats, in one of which the ectopic template was in the other parental strand and in the other of which the ectopic template was in the daughter strand itself. Examination of other mutations added imperfect direct repeats as mutagenic templates (Ripley et al. 1986;Shinedling et al. 1987). Templated mutations are usually invoked when they simultaneously acquire multiple changes for which a plausible donor template can be found. Despite the intrinsic fascination of templated complex mutations and their potential importance for certain evolutionary paths and some human genetic disorders, the enzymology that creates them has not been characterized.Template switching also occurs in a mutation-avoiding mode called replication repair. In the canonical model for replication repair (Fujiwara and Tatsumi 1976;Higgins et al. 1976), a primer strand whose extension has been blocked by DNA damage switches to the other daughter strand as a template, extends briefly, and then switches back to its cognate template, thus accurately bypassing the damaged site.In 1987, a mechanism for su...

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“…Cite this article as Cold Spring Harb Perspect Biol 2015;7:a016444 comitant displacement of Gp32 from ssDNA (Liu et al 2006(Liu et al , 2013. The yeast Rad52 protein promotes a similar exchange of Rad51/RPA proteins on ssDNA during presynapsis , and this mechanism likely applies to all RMPs of this subclass.…”

Section: Dna Pairing and Annealingmentioning

confidence: 99%

“…This reflects the requirement for ATP binding to induce the extended, active form of the presynaptic filament. On the other hand, ATP hydrolysis appears to regulate filament activity and to provide for filament turnover or dynamic instability (Kowalczykowski 1991;Cox 2003;Liu et al 2006Liu et al , 2011a. In addition, DNA-pairing protein-promoted pairing reactions are stimulated by ssDNA-binding and recombination mediator proteins, which are described below (see Daley et al 2014;Zelensky et al 2014).…”

Section: Properties Of Dna-pairing Proteinsmentioning

confidence: 99%

“…SSBs stimulate these reactions by at least two different mechanisms: First, SSBs facilitate presynaptic filament assembly on ssDNA. The SSB removes secondary structure from ssDNA, optimizing its conformation for handoff to the DNA-pairing protein, a transaction that typically requires a cognate recombination mediator protein in addition to DNA-pairing protein and ATP (Umezu et al 1993;Sung 1997b;Sugiyama and Kowalczykowski 2002;Liu et al 2006) (see below). Second, SSBs stabilize the D-loop product of the pairing reaction by sequestering the displaced strand (Kodadek 1990).…”

Section: Properties Of Dna-pairing Proteinsmentioning

confidence: 99%

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DNA-Pairing and Annealing Processes in Homologous Recombination and Homology-Directed Repair

Morrical

2015

Cold Spring Harb Perspect Biol

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114

127

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The formation of heteroduplex DNA is a central step in the exchange of DNA sequences via homologous recombination, and in the accurate repair of broken chromosomes via homology-directed repair pathways. In cells, heteroduplex DNA largely arises through the activities of recombination proteins that promote DNA-pairing and annealing reactions. Classes of proteins involved in pairing and annealing include RecA-family DNA-pairing proteins, single-stranded DNA (ssDNA)-binding proteins, recombination mediator proteins, annealing proteins, and nucleases. This review explores the properties of these pairing and annealing proteins, and highlights their roles in complex recombination processes including the double Holliday junction (DhJ) formation, synthesis-dependent strand annealing, and singlestrand annealing pathways-DNA transactions that are critical both for genome stability in individual organisms and for the evolution of species.

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Dynamics of Bacteriophage T4 Presynaptic Filament Assembly from Extrinsic Fluorescence Measurements of Gp32-Single-stranded DNA Interactions

Cited by 29 publications

References 54 publications

Structure and mechanism of the phage T4 recombination mediator protein UvsY

Structure and mechanism of the phage T4 recombination mediator protein UvsY

Templated Mutagenesis in Bacteriophage T4 Involving Imperfect Direct or Indirect Sequence Repeats

DNA-Pairing and Annealing Processes in Homologous Recombination and Homology-Directed Repair

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