Multiple C-Terminal Tails within a Single \u3cem\u3eE. coli\u3c/em\u3e SSB Homotetramer Coordinate DNA Replication and Repair

Escherichia coli single-stranded DNA binding protein (SSB) plays essential roles in DNA replication, recombination and repair. SSB functions as a homotetramer with each subunit possessing a DNA binding domain (OB-fold) and an intrinsically disordered C-terminus, of which the last nine amino acids provide the site for interaction with at least a dozen other proteins that function in DNA metabolism. To examine how many C-termini are needed for SSB function, we engineered covalently linked forms of SSB that possess only one or two C-termini within a four-OB-fold “tetramer”. Whereas E. coli expressing SSB with only two tails can survive, expression of a single-tailed SSB is dominant lethal. E. coli expressing only the two-tailed SSB recovers faster from exposure to DNA damaging agents but accumulates more mutations. A single-tailed SSB shows defects in coupled leading and lagging strand DNA replication and does not support replication restart in vitro. These deficiencies in vitro provide a plausible explanation for the lethality observed in vivo. These results indicate that a single SSB tetramer must interact simultaneously with multiple protein partners during some essential roles in genome maintenance

The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans.

Crossing over between homologs is initiated in meiotic prophase by the formation of DNA double-strand breaks that occur throughout the genome. In the major interference-responsive crossover pathway in baker's yeast, these breaks are resected to form 3' single-strand tails that participate in a homology search, ultimately forming double Holliday junctions (dHJs) that primarily include both homologs. These dHJs are resolved by endonuclease activity to form exclusively crossovers, which are critical for proper homolog segregation in Meiosis I. Recent genetic, biochemical, and molecular studies in yeast are consistent with the hypothesis of Mlh1-Mlh3 DNA mismatch repair complex acting as the major endonuclease activity that resolves dHJs into crossovers. However, the mechanism by which the Mlh1-Mlh3 endonuclease is activated is unknown. Here, we provide evidence that Mlh1-Mlh3 does not behave like a structure-specific endonuclease but forms polymers required to generate nicks in DNA. This conclusion is supported by DNA binding studies performed with different-sized substrates that contain or lack polymerization barriers and endonuclease assays performed with varying ratios of endonuclease-deficient and endonuclease-proficient Mlh1-Mlh3. In addition, Mlh1-Mlh3 can generate religatable double-strand breaks and form an active nucleoprotein complex that can nick DNA substrates in trans. Together these observations argue that Mlh1-Mlh3 may not act like a canonical, RuvC-like Holliday junction resolvase and support a novel model in which Mlh1-Mlh3 is loaded onto DNA to form an activated polymer that cleaves DNA

The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans

Crossing over between homologs is initiated in meiotic prophase by the formation of DNA double-strand breaks that occur throughout the genome. In the major interference-responsive crossover pathway in baker’s yeast, these breaks are resected to form 3' single-strand tails that participate in a homology search, ultimately forming double Holliday junctions (dHJs) that primarily include both homologs. These dHJs are resolved by endonuclease activity to form exclusively crossovers, which are critical for proper homolog segregation in Meiosis I. Recent genetic, biochemical, and molecular studies in yeast are consistent with the hypothesis of Mlh1-Mlh3 DNA mismatch repair complex acting as the major endonuclease activity that resolves dHJs into crossovers. However, the mechanism by which the Mlh1-Mlh3 endonuclease is activated is unknown. Here, we provide evidence that Mlh1-Mlh3 does not behave like a structure-specific endonuclease but forms polymers required to generate nicks in DNA. This conclusion is supported by DNA binding studies performed with different-sized substrates that contain or lack polymerization barriers and endonuclease assays performed with varying ratios of endonuclease-deficient and endonuclease-proficient Mlh1-Mlh3. In addition, Mlh1-Mlh3 can generate religatable double-strand breaks and form an active nucleoprotein complex that can nick DNA substrates in trans. Together these observations argue that Mlh1-Mlh3 may not act like a canonical, RuvC-like Holliday junction resolvase and support a novel model in which Mlh1-Mlh3 is loaded onto DNA to form an activated polymer that cleaves DNA

Model for Mlh1-Mlh3 activation.

<p>(A) Model for Mlh1-Mlh3’s (blue and purple heterodimer) endonuclease activation dependent on polymerization of the protein. (1) Mlh1-Mlh3 binds DNA and forms a polymer with a specified polarity. (2) Polymer formation proceeds and if other factors are present, they are displaced. (3) A polymer is formed of the critical length and stability for activation. (4) The activated polymer can introduce a nick into one strand of the duplex DNA. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001164#sec002" target="_blank">Results</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001164#sec007" target="_blank">Discussion</a> for details. (B) Mlh1-Mlh3 activation in the context of in vitro substrates. Red arrow indicates nicking activity by an active polymer. (1) An Mlh1-Mlh3 polymer can form on circular or linear DNA. For linear DNA, the molecule must be a length critical for polymer stability. (2) On circular substrate that is at least 12 kb, Mlh1-Mlh3 binds to adjacent regions of duplex DNA, leading to DSB formation. (3) Top: on circular substrates with preexisting nicks, polymers form and interactions between polymers direct Mlh1-Mlh3 to nick opposite the preexisting nick. Bottom: nicking in trans. If the adjacent regions of dsDNA do not have the same polarity, interactions between polymers may occur with a different orientation, promoting nicking to the duplex that does not contain a preexisting nick. (C) Hypothesis for how in vitro observations can be applied to understand HJ resolution. Bound Msh4-Msh5 recruits Mlh1-Mlh3 to form a polymer. Polymer formation and relative polarities of the duplex arms activate endonuclease activity to cleave strands with the same polarity. Branch migration could position nicks directly at the junction. (D) Hypothesis for how Mlh1-Mlh3 may resolve dHJs in vivo if the dHJ is unligated. The mode of resolution is different depending on where synthesis terminates. The grey square with a white question mark indicates unknown occupancy of one of the junctions. Mlh1-Mlh3 is recruited by Msh4-Msh5, which is then displaced by the Mlh1-Mlh3 polymer. The relative polarities of the two duplexes and the nicks resulting from DNA synthesis direct asymmetric resolution with respect to the two junctions. Cleavage could take place near the junction or some distance away. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001164#sec007" target="_blank">Discussion</a> for additional details. (E) Two possible conformations of a dHJ. Small black arrows in the top of the panel indicate where nicks would need to form to produce the CO products in the bottom of the panel. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001164#sec007" target="_blank">Discussion</a>. (F) Hypothesis for how Mlh1-Mlh3 may resolve type II dHJs in E. Mlh1-Mlh3 is recruited by Msh4-Msh5, which could be displaced by Mlh1-Mlh3. Polymer formation activates the endonuclease to cleave. Cleavage in this model can occur symmetrically with respect to both junctions, either near the junction or at a site away from the branch point.</p

Mlh1-Mlh3 binding to mismatched and Holliday junction substrates inhibit its endonuclease activity.

<p>In native agarose gels, migration of nicked product (n), linear product (black triangle), and closed circular substrate (cc) are indicated. All endonuclease reactions were carried out for 60 min, then stopped by addition of sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), and ProteinaseK as described in the Materials and methods. (A) Endonuclease activity was performed with supercoiled pUC18 as described in the Materials and methods. Where + Mg²⁺ or + Mn²⁺ is indicated, 1 mM MgCl₂ or MnSO₄ was added. Where both Mg²⁺ and Mn²⁺ are indicated, 0.5 mM of each was included. Where + Mlh1-Mlh3 is indicated, 300 nM wild type or Mlh1-mlh3D523N was added. (B) Mlh1-Mlh3 endonuclease activity on a 2.7 kb circular DNA substrate is inhibited by preincubating Mlh1-Mlh3 with oligonucleotide substrates. Mlh1-Mlh3 (100 nM) was preincubated with increasing amounts of ~50 bp double-stranded oligonucleotide substrates for 15 min at 30°C (0–2,000 nM): either homoduplex (0 μM, 10 μM, 25 μM, 50 μM, 100 μM, or 200 μM, expressed as total nucleotide concentration), +8 loop (0 μM, 10 μM, 25 μM, 50 μM, 100 μM, or 200 μM, expressed as total nucleotide concentration), or 30 bp armed Holliday junction (0 μM, 24 μM, 60 μM, 120 μM, 240 μM, or 480 μM, expressed as total nucleotide concentration). After the preincubation step, reactions were challenged with ~18 μM (expressed as total nucleotide concentration) 2.7 kb circular substrate and incubated by conditions described for endonuclease assays in the Materials and methods and analyzed by agarose gel. All lanes contain 1 mM Mg²⁺. (C) Average of quantification of plasmid nicked for four separate experiments from B; error bars represent standard deviation.</p

Negative stain images of Mlh1-Mlh3 binding to circular DNA using a K2 direct detector.

<p>(A) Representative electron micrograph of sample containing 3.6 nM of 2.7 kb circular DNA in the absence of protein. DNA is indicated by black arrow. A total 50 particles were assessed and all were classified as naked DNA particles. The inset area contains a zoomed view of one of the DNA molecules. The image for this molecule is 2-fold magnified with respect to the entire micrograph in this panel. (B) The electron micrograph shows a sample containing 300 nM Mlh1-Mlh3 in the absence of DNA. (C) Representative electron micrograph of sample containing 30 nM Mlh1-Mlh3 + 3.6 nM 2.7 kb circular DNA. Protein and DNA form both loosely (white arrows) and tightly packed (black arrow) clusters. Naked DNA was also observed in this sample but it is not shown in the electron micrograph. A total of 105 particles were analyzed under these conditions. 50 (48%) were completely condensed clusters, 22 (21%) were partially condensed, and 33 (31%) were naked DNA. (D) Electron micrograph showing sample containing 300 nM Mlh1-Mlh3 + 3.6 nM 2.7 kb circular DNA. Higher concentration of protein induces formation of more condensed protein–DNA clusters (black arrows). Mlh1-Mlh3 protein complexes are also seen in the background of the micrograph. 53 total particles were assessed. Of these, 50 (94%) were completely condensed clusters, 1 (2%) was a partially condensed, and 2 (4%) were naked DNA.</p

An activated Mlh1-Mlh3-DNA complex can nick DNA in trans.

<p>0.7 nM 7.2 kb closed circular M13mp18 phagemid and 1.8 nM 2.7 kb linear pUC18 substrate were incubated with 300 nM Mlh1-Mlh3 under standard endonuclease assay conditions either in isolation or within the same reaction as indicated. (A) Reaction products were run on an alkaline agarose gel. The 7.2 kb substrate was linearized with <i>Hin</i>dIII prior to loading in the alkaline agarose gel. The material in lane 4 and 5 was mixed and run as a control in lane 11 to demonstrate the readout for a negative result. The fraction of DNA nicked was measured by determining the band density in either the 7.2 kb linear band or the 2.7 kb linear band by subtracting the density in a region immediately above the band as background and comparing it to the band densities in the negative controls. (B) Prior to linearization with <i>Hin</i>dIII, 10 μL of each reaction was removed and run on a native agarose gel.</p

Preexisting nicks act as preferential nicking (but not loading) sites for Mlh1-Mlh3.

<p>(A) Mlh1-Mlh3 creates approximately the same amount of linear product regardless of how many preexisting nicks are in the circular substrate. 300 nM yeast Mlh1-Mlh3 on either closed circular 2.7 kb plasmid substrate (0 preexisting nicks), <i>Nt</i>.<i>Bsp</i>QI-treated substrate (one preexisting nick), <i>Nt</i>.<i>Bst</i>NBI-treated substrate (four preexisting nicks), or <i>Nt</i>.<i>Alw</i>I-treated substrate (ten preexisting nicks). Nicked (n) and linear (black triangle) products are shown. (B) Wild-type Mlh1-Mlh3 (300 nM) creates linear product on either closed circular 2.7 kb plasmid substrate or <i>Nt</i>.<i>Bst</i>NBI-treated substrate, while Mlh1-mlh3D523N (300 nM) is inactive on both. (C) Mapping the formation of Mlh1-Mlh3–induced nick opposite a preexisting nick. Top: experimental setup. Plasmid was either linearized with <i>Sap</i>I, which creates a 3-bp overhang, or substrate with a preexisting nick generated by <i>Nt</i>.<i>Bsp</i>QI was linearized by Mlh1-Mlh3. For each, linear product was gel extracted and annealed to a primer either complementary to the strand with the preexisting nick (primer A) or to the opposite strand (primer B). Primers were extended by T4 polymerase where indicated. Primer extension products were resolved by denaturing PAGE. For the <i>Sap</i>I linear control, extension of primer A gives a 60-mer and extension of primer B gives a 63-mer product. Bottom: lanes 1, 6, and 11 are radiolabeled 60-mer used as a primer extension marker. Lanes 2–5 show primer extension for the linear control, while lanes 7–10 show primer extension for Mlh1-Mlh3 linear product. Lane 12 is a duplicate of the material in lane 5.</p