SSB Protein Limits RecOR Binding onto Single-stranded DNA

Hobbs, Michael D.; Sakai, Akiko; Cox, Michael M.

doi:10.1074/jbc.m611007200

Cited by 80 publications

(99 citation statements)

References 79 publications

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“…interacts with the C-terminal tail of SSB (36,37). To further test the existence and importance of this interaction in the strand displacement reaction, we replaced wild-type SSB with two C-terminal SSB proteins that had 8 and 42 amino acids deleted from their C termini (30,31). We observed that neither supported strand displacement (Fig.…”

Section: Resultsmentioning

confidence: 89%

Strand Displacement by DNA Polymerase III Occurs through a τ-ψ-χ Link to Single-stranded DNA-binding Protein Coating the Lagging Strand Template

Yuan

McHenry

2009

Journal of Biological Chemistry

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In addition to the well characterized processive replication reaction catalyzed by the DNA polymerase III holoenzyme on single-stranded DNA templates, the enzyme possesses an intrinsic strand displacement activity on flapped templates. The strand displacement activity is distinguished from the singlestranded DNA-templated reaction by a high dependence upon single-stranded DNA binding protein and an inability of ␥-complex to support the reaction in the absence of . However, if ␥-complex is present to load ␤ 2 , a truncated protein containing only domains III-V will suffice. This truncated protein is sufficient to bind both the ␣ subunit of DNA polymerase (Pol) III and . This is reminiscent of the minimal requirements for Pol III to replicate short single-stranded DNA-binding protein (SSB)-coated templates where is only required to serve as a scaffold to hold Pol III and in the same complex (Glover, B., and McHenry, C. (1998) J. Biol. Chem. 273, 23476 -23484). We propose a model in which strand displacement by DNA polymerase III holoenzyme depends upon a Pol III----SSB binding network, where SSB is bound to the displaced strand, stabilizing the Pol III-template interaction. The same interaction network is probably important for stabilizing the leading strand polymerase interactions with authentic replication forks. The specificity constant (k cat /K m ) for the strand displacement reaction is ϳ300-fold less favorable than reactions on singlestranded templates and proceeds with a slower rate (150 nucleotides/s) and only moderate processivity (ϳ300 nucleotides). PriA, the initiator of replication restart on collapsed or misassembled replication forks, blocks the strand displacement reaction, even if added to an ongoing reaction.All cellular replicases are tripartite assemblies, consisting of a replicative polymerase (Pol 2 III in bacteria, Pol ␦ or ⑀ in eukaryotes), a sliding clamp processivity factor (␤ 2 in bacteria, proliferating cell nuclear antigen in eukaryotes), and a clamp loader composed of a five-protein core of AAA ϩ -like proteins that assembles the sliding clamp around DNA (DnaX complex in bacteria, RFC in eukaryotes) (1-3). The Escherichia coli DnaX complex comprises three copies of DnaX and one each of ␦, ␦Ј, and (4 -6). E. coli and many other bacteria contain two forms of DnaX, the full-length translation product and a shorter protein, ␥, that results from translational frameshifting (7-9). Both and ␥ contain the three domains that are required for ATP-dependent ␤ 2 loading onto DNA (6). The third domain of and ␥ is responsible for binding other DnaX subunits as well as ␦, ␦Ј, and (5, 10, 11). contains two additional domains that interact with the DnaB replicative helicase (domain IV) and Pol III (domain V) (12, 13).The primary determinant of processivity of the E. coli replicase is the interaction of Pol III with ␤ 2 (14, 15). However, other interactions stabilize the interaction of Pol III with the replication fork. Two protomers bind the DnaB helicase, further stabilizing the replicase at the fork...

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Section: Resultsmentioning

confidence: 89%

Strand Displacement by DNA Polymerase III Occurs through a τ-ψ-χ Link to Single-stranded DNA-binding Protein Coating the Lagging Strand Template

Yuan

McHenry

2009

Journal of Biological Chemistry

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“…RecA (d)ATP Hydrolysis Assays-The ssDNA-dependent dATP or ATP (denoted as (d)ATP) hydrolysis activity of RecA protein was performed as described (6,24). RecA-mediated (d)ATP hydrolysis was not affected within the first 180 min of incubation, even upon binding a silicon nanowire transistor (25).…”

Section: Methodsmentioning

confidence: 99%

“…As previously reported (24,26), the lag time, which represents the delay in reaction progress relative to a theoretically reaction curve lacking it, was derived from the time intercept of a linear regression line fit to the steady state portion of data in (d)ATP hydrolysis assays.…”

Section: Methodsmentioning

confidence: 99%

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Roles of Bacillus subtilis DprA and SsbA in RecA-mediated Genetic Recombination

Yadav

Carrasco

Serrano

et al. 2014

Journal of Biological Chemistry

102

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“…C-terminally deleted variants of SSB, in which the C-terminal domain (up to 42 residues) has been removed, bind somewhat more tightly to ssDNA than does wild type SSB (77,78). In trials to date, no ssDNA binding deficiency (relative to wild type SSB) has been detected with the SSB⌬C8 variant (76,79). Four of the final eight residues of E. coli SSB are aspartate residues, and many proteins interact with this acidic SSB C terminus, (73, 79 -91).…”

Section: Deleting 8 Amino Acid Residues From the Ssb C Terminus Decrementioning

confidence: 99%

SSB Antagonizes RecX-RecA Interaction

Baitin

Gruenig

Cox

2008

Journal of Biological Chemistry

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The bacterial RecA protein is the prototype of a nearly ubiquitous class of recombinase proteins (1-11). These enzymes promote DNA strand exchange reactions that lie at the heart of all recombination and recombinational DNA repair processes (11-16). The recombinase forms a right-handed helical filament, usually on single-stranded DNA (ssDNA).2 A homologous duplex DNA is then aligned with this bound single strand, and one strand (the one complementary to the bound single strand) is transferred from the duplex substrate to form a new duplex within the filament. This process is used to reconstruct stalled replication forks, repair double strand breaks, facilitate meiotic cross-overs in eukaryotes, and facilitate genetic exchanges of many types in almost every organism. Typical reactions are illustrated in Fig. 1.The assembly and disassembly of RecA protein filaments on DNA has been studied in some detail, although understanding is not complete. Nucleation occurs most readily on ssDNA. There are discrete nucleation and extension phases in the assembly process, with extension proceeding primarily 5Ј to 3Ј relative to the ssDNA (17, 18). Disassembly also proceeds primarily 5Ј to 3Ј, with monomeric subunits being added on one end and subtracted from the other (19,20). Dissociation at the disassembly end is coupled to ATP hydrolysis (21,22).RecA-promoted reactions are important to cell viability. Escherichia coli strains lacking a functional recA gene are viable but highly sensitive to DNA-damaging agents (23-25). Recombinational DNA repair is thus important in bacteria, especially for the repair of stalled or collapsed replication forks (26 -30). However, recombination can also have deleterious consequences and must be regulated. Recombination between repeated sequences in the genome, for example, could result in the deletion of essential genes.In the past decade, multiple new RecA regulatory mechanisms have been elucidated. The expression of the recA gene is regulated as part of the SOS response (31-35). The native RecA protein is auto-regulated by the 17 amino acid residues at its own C terminus, because virtually every RecA activity is enhanced when those 17 amino acids are deleted (36 -41). Finally, RecA function is regulated by a growing array of regulatory proteins.There are at least eight proteins in a typical E. coli cell that modulate RecA protein function to some extent. Much of the regulation is focused on RecA filament assembly and disassembly. One modulator of RecA filament dynamics is SSB. SSB strongly inhibits the nucleation phase of RecA filament assembly (42) but stimulates the extension phase, removing secondary structure in ssDNA that would otherwise impede filament growth (43-46). Although ordinarily SSB protein prevents filament nucleation, single RecA monomers can easily be added to an existing filament and displace SSB from DNA at the rate of filament extension (47).Additional proteins affect other aspects of RecA protein filament function, often by interacting directly with RecA. The nucleation ...

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SSB Protein Limits RecOR Binding onto Single-stranded DNA

Cited by 80 publications

References 79 publications

Strand Displacement by DNA Polymerase III Occurs through a τ-ψ-χ Link to Single-stranded DNA-binding Protein Coating the Lagging Strand Template

Strand Displacement by DNA Polymerase III Occurs through a τ-ψ-χ Link to Single-stranded DNA-binding Protein Coating the Lagging Strand Template

Roles of Bacillus subtilis DprA and SsbA in RecA-mediated Genetic Recombination

SSB Antagonizes RecX-RecA Interaction

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