Drosophila Muller F Elements Maintain a Distinct Set of Genomic Properties Over 40 Million Years of Evolution

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25–50%) than euchromatic reference regions (3–11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11–27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4–3.6 vs. 8.4–8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu

DYNAMICS OF THE MISMATCH REPAIR COMPLEXES DURING DNA REPLICATION

DNA mismatch repair (MMR) functions mainly to correct mispaired bases that escape the proofreading activity of the DNA polymerase during replication. Defects in MMR genes have been linked to compromised genome stability and diseases including cancer. MMR is a highly conserved process and the yeast Saccharomyces cerevisiae is an ideal model organism to explore aspects of MMR because of the ease of manipulation and homology to the human system. MMR initiates when a mismatch in the DNA helix is recognized by MutS homologs. Subsequent events include excision of the error-containing strand followed by re-synthesis. A critical step in this process is directing repair to the newly synthesized strand, which requires a strand discrimination signal. Current data suggest that transient discontinuities in the DNA backbone, known as nicks, generated during replication serve as the strand discrimination signal. Additionally, histones have the capacity to block mismatch recognition and are known to rapidly assemble behind the replication fork. Thus, there must be a short window of opportunity for the MutS homologs to scan for mismatches and access the strand discrimination signals during replication. To address these unresolved issues, we hypothesize that the MMR machinery tracks with the replisome to allow for efficient scanning and access to the strand discrimination signal. We employed chromatin immunoprecipitation and DNA tiling microarrays (ChIP-chip) to determine the distribution of the eukaryotic MutS complexes during replication. The data indicate that during S-phase of the cell cycle MutS binds origins of replication and shows bi-directional occupancy of regions flanking the origins over time with timing consistent with fork progression. Importantly, MutS displays the same origin binding and spreading pattern as the leading strand DNA polymerase over multiple experiments. In sum, our data supports the hypothesis of the MMR machinery tracking with the replisome. There are two MutS complexes that occur in eukaryotes, MutSα (Msh2/Msh6) and MutSβ (Msh2/Msh3). Both complexes recognize the different types of mismatches that arise during DNA replication. Reporter constructs have traditionally been utilized to assay the types of mismatches targeted by each complex. With the availability of new techniques, we can now analyze the functions of MutSα and MutSβ on a genome wide scale. In this work, we used next generation sequencing to determine the mutation spectra in strains lacking MSH2, MSH3 or MSH6. Our studies confirm the findings of previous genetic experiments that MutSα and MutSβ are functionally redundant for repair at HPRs; however, each complex is essential for ~6-7% of the mismatches generated during replication. In this work, we provide evidence that both complexes should be in the vicinity of the replisome to ensure that majority of the mutations can be avoided during replication

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

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

Polε dynamics during DNA replication.

<p>Analysis of Pol2 (Polε) dynamics during DNA replication using ChIP-chip. Each row corresponds to ChIP-chip signal at the indicated times at G1 or to the time point series taken during S phase (0 to 30 minutes or 0 to 50 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. Polε signal (green) 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 converge.</p

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 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 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 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