Mediator proteins orchestrate enzyme-ssDNA assembly during T4 recombination-dependent DNA replication and repair

Bleuit, Jill S.; Xu, Hang; Ma, Yujie; Wang, Tongsheng; Liu, Jie; Morrical, Scott W.

doi:10.1073/pnas.131007498

Cited by 79 publications

(96 citation statements)

References 34 publications

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“…Omitting the lagging strand template increased the yield of the 120-nt product (Fig. 3, lane 4) in accord with previous reports that T4 replication plus recombination proteins can catalyze conservative DNA replication (29,30). Replacing the 94-nt hybrid RNA-DNA lagging strand with a 90-nt DNA strand did not alter the efficiency of template switching (Fig.…”

Section: Resultssupporting

confidence: 91%

UvsX Recombinase and Dda Helicase Rescue Stalled Bacteriophage T4 DNA Replication Forks in Vitro

Kadyrov¹,

Drake

2004

Journal of Biological Chemistry

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The rescue of stalled replication forks via a series of steps that include fork regression, template switching, and fork restoration often has been proposed as a major mechanism for accurately bypassing non-coding DNA lesions. Bacteriophage T4 encodes almost all of the proteins required for its own DNA replication, recombination, and repair. Both recombination and recombination repair in T4 rely on UvsX, a RecA-like recombinase. We show here that UvsX plus the T4-encoded helicase Dda suffice to rescue stalled T4 replication forks in vitro. This rescue is based on two sequential template-switching reactions that allow DNA replication to bypass a non-coding DNA lesion in a non-mutagenic manner.DNA template-strand lesions that prevent the extension of primer strands are usually lethal. Four general mechanisms operate to overcome such lethality: direct repair that regenerates a native structure (e.g. photoreaction and dealkylation), excision repair that replaces damaged bases or deoxyribose residues, translesion synthesis that is usually mutation-prone, and recombination repair that accurately bypasses DNA damage.The essence of recombination repair is that it derives accurate genetic information from the nascent sister chromatid (or from a homologous chromosome when repairing a double strand break); excision repair, on the other hand, derives the requisite genetic information from the complementary strand of the same double helix. Recombination repair was first characterized in Escherichia coli (1-4). The classical E. coli breakreunion model holds that blocked primer extension is followed by downstream reinitiation, leaving a gap that is then filled at least in part by the physical donation of a strand of appropriate polarity cut from the sister chromatid. The donating strand gap is filled by DNA synthesis, whereas the blocking lesion remains as a problem for the future.Recent progress in understanding recombination repair, based mainly on work with E. coli, has highlighted its importance for rescuing replication forks stalled at sites of DNA damage or spontaneous fork collapse (8 -11). One model of recombination repair (Fig. 1) invokes a topological rearrangement in which the fork regresses into a configuration in which the parental strands reanneal and the two daughter strands anneal. This rearrangement generates a structure that constitutes a Holliday junction and that to some resembles a chicken foot (12). In the case of a primer strand whose extension is blocked by a lesion in the template strand, fork regression provides a template that allows limited primer extension. Subsequent reformation of a conventional replication fork then provides a primer strand that has accurately bypassed the template lesion. This pathway was named replication repair and was initially suggested to occur during mammalian DNA replication (5). It was later validated by genetical and enzymological analyses in phage T4 (6, 7).In addition to a DNA polymerase, the canonical model of replication repair (Fig. 1) immediately suggests roles f...

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Section: Resultssupporting

confidence: 91%

UvsX Recombinase and Dda Helicase Rescue Stalled Bacteriophage T4 DNA Replication Forks in Vitro

Kadyrov¹,

Drake

2004

Journal of Biological Chemistry

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“…So-called ''mediator'' proteins appear to play this role. These proteins promote efficient formation of recombinase oligomers on ssDNA that are coated with ssb (34,35). The bacteriophage T4 UvsY was the first protein to be shown to have mediator activity in vitro (21,36,37).…”

Section: Reca-like Recombinases Assemble On Single-strand Dna (Ssdna)mentioning

confidence: 99%

“…Both the S. cerevisiae Rad52 and heterodimeric Rad55͞Rad57 have been shown to have mediator function in vitro (35,(49)(50)(51)(52). Both Rad52 and Rad55͞57 can overcome inhibition of Rad51-mediated strand exchange by RPA.…”

Section: Reca-like Recombinases Assemble On Single-strand Dna (Ssdna)mentioning

confidence: 99%

Assembly of RecA-like recombinases: Distinct roles for mediator proteins in mitosis and meiosis

Gasior

Olivares

Ear

et al. 2001

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

160

125

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Members of the RecA family of recombinases from bacteriophage T4, Escherichia coli, yeast, and higher eukaryotes function in recombination as higher-order oligomers assembled on tracts of single-strand DNA (ssDNA). Biochemical studies have shown that assembly of recombinase involves accessory factors. These studies have identified a class of proteins, called recombination mediator proteins, that act by promoting assembly of recombinase on ssDNA tracts that are bound by ssDNA-binding protein (ssb). In the absence of mediators, ssb inhibits recombination reactions by competing with recombinase for DNA-binding sites. Here we briefly review mediated recombinase assembly and present results of new in vivo experiments. Immuno-double-staining experiments in Saccharomyces cerevisiae suggest that Rad51, the eukaryotic recombinase, can assemble at or near sites containing ssb (replication protein A, RPA) during the response to DNA damage, consistent with a need for mediator activity. Correspondingly, mediator gene mutants display defects in Rad51 assembly after DNA damage and during meiosis, although the requirements for assembly are distinct in the two cases. In meiosis, both Rad52 and Rad55͞57 are required, whereas either Rad52 or Rad55͞57 is sufficient to promote assembly of Rad51 in irradiated mitotic cells. Rad52 promotes normal amounts of Rad51 assembly in the absence of Rad55 at 30°C but not 20°C, accounting for the cold sensitivity of rad55 null mutants. Finally, we show that assembly of Rad51 is induced by radiation during S phase but not during G1, consistent with the role of Rad51 in repairing the spontaneous damage that occurs during DNA replication.H omologous recombination reactions promote repair of DNA ends formed by double-strand breaks (DSBs) and by replication fork collapse. Recombinational repair also allows cells to replicate past DNA lesions that block the progress of DNA polymerase. In phage T4, recombination is critical for initiating replication. In eukaryotes, homologous recombination is critical for accurate reductional segregation of chromosomes during meiosis. Finally, in prokaryotes, recombination allows horizontal transfer of alleles among and between bacteria and phage.At the center of homologous recombination are the recombinases, proteins that promote the formation of heteroduplex DNA. Of particular importance are the recombinases of the RecA family, including RecA in eubacteria, RadA in archea, Rad51 and Dmc1 in eukaryea, and the bacteriophage T4 UvsX protein.RecA-Like Recombinases Assemble on Single-Strand DNA (ssDNA).

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“…In the case of gp41, which does not interact with gp32 directly, the cooperative binding of gp32 to the displaced strand inhibits gp41 loading, causing a lag time in the onset of rapid DNA replication (12). This lag time is eliminated by gp59, a mediator protein that loads gp41 onto gp32-saturated ssDNA molecules (12)(13)(14)(15)(16). gp59 also binds * This work was supported by National Institutes of Health Grants GM48847 (to S. W. M.) and GM42569 (to D. P. G.) and by an award from the Lake Champlain Cancer Research Organization (to S. W. M.).…”

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confidence: 99%

Dual Functions of Single-stranded DNA-binding Protein in Helicase Loading at the Bacteriophage T4 DNA Replication Fork

Wang

Villemain

et al. 2004

Journal of Biological Chemistry

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Semi-conservative DNA synthesis reactions catalyzed by the bacteriophage T4 DNA polymerase holoenzyme are initiated by a strand displacement mechanism requiring gp32, the T4 single-stranded DNA (ssDNA)-binding protein, to sequester the displaced strand. After initiation, DNA helicase acquisition by the nascent replication fork leads to a dramatic increase in the rate and processivity of leading strand DNA synthesis. In vitro studies have established that either of two T4-encoded DNA helicases, gp41 or dda, is capable of stimulating strand displacement synthesis. The acquisition of either helicase by the nascent replication fork is modulated by other protein components of the fork including gp32 and, in the case of the gp41 helicase, its mediator/ loading protein gp59. Here, we examine the relationships between gp32 and the gp41/gp59 and dda helicase systems, respectively, during T4 replication using altered forms of gp32 defective in either protein-protein or protein-ssDNA interactions. We show that optimal stimulation of DNA synthesis by gp41/gp59 helicase requires gp32-gp59 interactions and is strongly dependent on the stability of ssDNA binding by gp32. Fluorescence assays demonstrate that gp59 binds stoichiometrically to forked DNA molecules; however, gp59-forked DNA complexes are destabilized via protein-protein interactions with the C-terminal "A-domain" fragment of gp32. These and previously published results suggest a model in which a mobile gp59-gp32 cluster bound to lagging strand ssDNA is the target for gp41 helicase assembly. In contrast, stimulation of DNA synthesis by dda helicase requires direct gp32-dda protein-protein interactions and is relatively unaffected by mutations in gp32 that destabilize its ssDNA binding activity. The latter data support a model in which protein-protein interactions with gp32 maintain dda in a proper active state for translocation at the replication fork. The relationship between dda and gp32 proteins in T4 replication appears similar to the relationship observed between the UL9 helicase and ICP8 ssDNA-binding protein in herpesvirus replication.DNA helicase acquisition by a nascent DNA replication fork is essential for reconstituting rapid, processive DNA synthesis. This principle is illustrated dramatically by the bacteriophage T4 DNA replication system (1-2). T4 encodes two DNA helicases known to affect the movement and properties of DNA replication forks; the gene 41 protein (gp41) and the dda protein, respectively. gp41 is the essential replicative helicase of the T4 phage. This hexameric enzyme translocates processively in a 5Ј 3 3Ј direction on the displaced lagging strand of the dsDNA 1 template, thus enhancing the rate and processivity of leading strand DNA synthesis catalyzed by the T4 DNA polymerase holoenzyme (gp43, gp44/62, and gp45 proteins) (1-3). In addition, gp41 is an obligatory component of the T4 primosome (helicase/primase complex) and is, thus, essential for lagging strand DNA synthesis (4 -5). A second T4-encoded DNA helicase, dda protein, stimula...

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Mediator proteins orchestrate enzyme-ssDNA assembly during T4 recombination-dependent DNA replication and repair

Cited by 79 publications

References 34 publications

UvsX Recombinase and Dda Helicase Rescue Stalled Bacteriophage T4 DNA Replication Forks in Vitro

UvsX Recombinase and Dda Helicase Rescue Stalled Bacteriophage T4 DNA Replication Forks in Vitro

Assembly of RecA-like recombinases: Distinct roles for mediator proteins in mitosis and meiosis

Dual Functions of Single-stranded DNA-binding Protein in Helicase Loading at the Bacteriophage T4 DNA Replication Fork

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