MutSα and MutSβ both bind origins during replication.
(A) Msh2, Msh3, and Msh6 levels are consistent with equal ratios of MutSα and MutSβ in the cell. 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) MutSα tracks with the replisome. Cells were processed for ChIP-chip as described above. An example of binding of Msh6 and Polε at ARS1407 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. (C) MutSβ binds ARS305 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. (D) Msh2 binding of ARS305 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.