The Eukaryotic Mismatch Recognition Complexes Track with the Replisome during DNA Synthesis

<div><p>During replication, mismatch repair proteins recognize and repair mispaired bases that escape the proofreading activity of DNA polymerase. In this work, we tested the model that the eukaryotic mismatch recognition complex tracks with the advancing replisome. Using yeast, we examined the dynamics during replication of the leading strand polymerase Polε using Pol2 and the eukaryotic mismatch recognition complex using Msh2, the invariant protein involved in mismatch recognition. Specifically, we synchronized cells and processed samples using chromatin immunoprecipitation combined with custom DNA tiling arrays (ChIP-chip). The Polε signal was not detectable in G1, but was observed at active origins and replicating DNA throughout S-phase. The Polε signal provided the resolution to track origin firing timing and efficiencies as well as replisome progression rates. By detecting Polε and Msh2 dynamics within the same strain, we established that the mismatch recognition complex binds origins and spreads to adjacent regions with the replisome. In mismatch repair defective PCNA mutants, we observed that Msh2 binds to regions of replicating DNA, but the distribution and dynamics are altered, suggesting that PCNA is not the sole determinant for the mismatch recognition complex association with replicating regions, but may influence the dynamics of movement. Using biochemical and genomic methods, we provide evidence that both MutS complexes are in the vicinity of the replisome to efficiently repair the entire spectrum of mutations during replication. Our data supports the model that the proximity of MutSα/β to the replisome for the efficient repair of the newly synthesized strand before chromatin reassembles.</p></div

MutSα and MutSβ both bind origins during replication.

<p><b>(A) Msh2, Msh3, and Msh6 levels are consistent with equal ratios of MutSα and MutSβ in the cell</b>. Cultures were grown to mid-exponential phase and proteins were extracted and detected by immunoblotting. The proteins were detected using antibodies for the myc epitope. Lane 1: contains Msh2-myc tagged extracts. Lane 2: all three components of the MutS complexes are myc-tagged (Msh2-myc, Msh6-myc, and Msh3-myc). The loading control was visualized using α-Kar2 antibody. The bands were quantified using image J software. <b>(B)</b> MutSα tracks with the replisome. Cells were processed for ChIP-chip as described above. An example of binding of Msh6 and Polε at <i>ARS1407</i> is shown. The log₂ (ChIP/Input) were visualized as using the Integrated Genome Browser and the y-axis is set at 3 (or ~8 fold maximum) for each row. Msh6 (red-brown), Polε (green), no tag (black) and Mcm4 (purple) signals are included. <b>(C)</b> MutSβ binds <i>ARS305</i> during S Phase. Samples were prepared for ChIP as described above. The DNA was quantified by PCR (qPCR) to ensure that a ChIP-specific signal was detectable. Three technical replicates were performed for each time point. Samples were amplified and the threshold cycles (Ct) were determined using the Sequence Detection System, SDS version 2.3 software (Applied Biosystems). ChIP DNA samples for Polε (green), Msh3 (red-brown), no tag (black) and Mcm4 (purple) as well as input DNA at three dilutions were quantitied using pPCR. The error bars represent standard error of the mean. <b>(D)</b> Msh2 binding of <i>ARS305</i> during S Phase. Samples were prepared and analyzed using ChIP-PCR as described above for Panel C. ChIP DNA samples for Polε (green), Msh2 (blue), no tag (black) and Mcm4 (purple) as well as input DNA at three dilutions were quantitied. The error bars represent standard error of the mean.</p

Msh2 dynamics during DNA replication.

<p>Time course ChIP-chip experiment for Msh2-myc (Msh2). Each row corresponds to ChIP-chip signal during G1 or to the time point series taken during S phase (0 to 30 minutes). The tiling array data were visualized using the Integrated Genome Browser program (Affymetrix) and are depicted as peaks correspond to log2 ratios (ChIP/Input). The y-axis is set to 2.5 (or a ~6-fold maximum signal). Black bars below the data denote position of origins in the genome databases. The red bars represent origins not found in the genome database. Chromosomal coordinates represent x 10³ kb. Mcm4 (Mcm) signal, shown in purple, is visible at potential origins during G1 and non-specific signals shown in black are detected in the no tag control IP during G1. Msh2 signal (blue) is detected at active origins. Representative regions are shown including: <b>(A)</b> active origins (<i>ARS305</i> and <i>ARS306</i>) and adjacent inactive origins (<i>ARS301</i>, <i>ARS303</i>, <i>ARS304</i> and <i>ARS320</i>), <b>(B)</b> an early-efficient origin (<i>ARS315</i>), <b>(C)</b> adjacent early-efficient (<i>ARS607</i>), early-inefficient, (<i>ARS608</i>), late-inefficient, <i>ARS609</i>, <b>(D)</b> early-efficient origins (<i>ARS1207</i> and <i>ARS1209</i>) flanking an inactive origin (<i>ARS1208</i>), <b>(E)</b> a ~100 kb region of chromosome IV where the advancing forks from <i>ARS413</i> and <i>ARS414</i> are observed.</p

Msh2 and Polε dynamics are similar during DNA replication.

<p>Cells were fixed for 45 minutes, the samples were divided and ChIP was performed with specified antibodies to detect Polε-HA (green) and Msh2-myc (blue). The distribution was visualized using the Integrated Genome Browser program (Affymetrix) as log₂ ratios (ChIP/Input) with the scale set at 2.5 (~ 6 fold increase) for all samples. Each row corresponds to ChIP-chip signal during G1 or to the time point series taken during S phase (0–50 min). Black bars below the data denote position of origins in the genome databases. Chromosomal coordinates represent x 10³ kb. Mcm4 (Mcm4) signal, shown in purple, is visible at potential origins during G1 and non-specific signals shown in black are detected in the no tag control IP during G1. Representative regions are shown including: <b>(A)</b><i>ARS1407</i>, where there is an initial unidirectional distribution of signal that is followed by bi-directional progression at later time point, and <b>(B)</b> the early-efficient <i>ARS1012</i> and the early-inefficient <i>ARS1013</i>.</p

Msh2 and Polε exhibit co-incident signal during S phase.

<p>Three independently performed experiments were used to calculate <i>p</i>-values. The samples were analyzed at the time of arrest (G1) and six additional time points during S phase for Polε and Msh2. The data are visualized as the negative of the log₁₀ of the calculated <i>p-</i>values using the Integrated Genome Browser for Mcm4 (Mcm, purple), no tag (black), Msh2 (blue) and Pol2 (Polε, green). Because the Mcm4 signal is so significant, the histogram is scaled to 27 (or reflecting a <i>p-</i>value ~10⁻²⁷ for the most signal values). The Msh2 and the “no tag” control graphs are set to 10 and the Polε graphs to 20. Black bars below the data denote position of origins in the genome databases. The red bars represent origins not found in the genome database. Chromosomal coordinates represent x10³ kb. Representative regions are shown including: <b>(A)</b> early-efficient origins (<i>ARS1207</i> and <i>ARS1209</i>) flanking an inactive origin (<i>ARS1208</i>), <b>(B)</b><i>ARS1213</i>, and <b>(C)</b><i>ARS416</i>.</p

Msh2 displays aberrant binding at origins in a double mutant strain that disrupts the interaction between MutSα and PCNA.

<p><b>(A)</b> Double mutant cells (<i>msh6-F33A</i>,<i>F34A pol30-204</i>) expressing the Msh6^PIP and Pol30^C81R were analyzed at the time of arrest (G1) and additional time points 10 minutes apart during S phase for Polε and Msh2. The log₂ (ChIP/Input) were visualized as using the Integrated Genome Browser for Mcm4 (purple), no tag (black), Msh2 (blue) and Polε (green). The graphs were set to 2.0 for all data (~6 fold maximum increase). The black bar below the data denotes the position of <i>ARS315</i>. The same region for wild-type is shown for comparison for Pol2 (B) and Msh2 (C). The images in panes B and C are also shown in Figs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005719#pgen.1005719.g001" target="_blank">1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005719#pgen.1005719.g002" target="_blank">2</a>.</p

Model for MutS mismatch recognition during replication.

<p>The model described in the text is depicted above with schematics of MutS complexes (green and red) with a flexible tether (purple); DNA polymerase (multi-subunit complexes shown in light blue and grey); PCNA (dark blue circles), unmodified histones (green circles); modified histones (green circles with blue tag); single stranded binding proteins (orange circles); DNA polymerase alpha (multi-subunit complex shown in purple circles); and the Mcm4 helicase (red). The template DNA is shown with black lines and the newly synthesized DNA with green lines. The direction of polymerization of the DNA is shown with arrowheads.</p

Msh2 and Polε co-localize to origins during S phase in a strain expressing a PCNA/Pol30 MMR defective variant, Pol30^C22Y.

<p>The samples were analyzed at the time of arrest (G1) and two additional time points in S phase, 10 minutes apart (20 min and 30 min) for Polε and Msh2. The log₂ (ChIP/Input) were visualized as using the Integrated Genome Browser for Mcm4 (Mcm, purple), no tag (black), Msh2 (blue) and Pol2 (Polε, green). The graphs were set to 2.5 for all data (~6 fold maximum increase). Black bars below the data denote position of origins in the genome databases. Chromosomal coordinates represent x10³ kb. Representative regions are shown including: <b>(A)</b><i>ARS315</i><b>(B)</b><i>ARS51</i>.</p