Unknown,Transcriptomics,Genomics,Proteomics

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Interrelationships between replication infidelity and genome composition

ABSTRACT: An enduring issue in evolutionary and cancer biology is how replication infidelity influences genome composition and vice versa. Here we examine this issue by sequencing the genomes of diploid budding yeast strains that are either mismatch repair (MMR) proficient or deficient and encode wild type or mutator variants of the three major nuclear DNA replicases. Analysis of over 43,000 mutations that accumulated in the absence of selective pressure demonstrates that the nuclear DNA replication machinery generates less than one mismatch per genome and in combination with MMR, achieves a genome-wide per base error rate of 1.7 x 10-10. Absent both MMR and purifying selection, replication error patterns strongly depend on replication origin proximity, replication fork direction, and the local DNA sequence. Preferred sequences were observed for base substitutions and deletions. Error rates also vary with replication time, in linker versus nucleosome-bound DNA, in 5'- and 3'-untranslated regions, in coding regions and in intergenic DNA. This genome-wide view shows that replication fidelity is amazingly high but heterogeneous, in patterns that suggest the underlying mechanisms by which replication modulates genome stability and composition and vice versa. Six to ten isolates were sequenced for each combination of DNA polymerase (WT, pol1-L868M, pol2-M644G, pol3-L612M) and mismatch repair (proficient, deficient) genotypes. A single WT isolate was sequenced following micrococcal nuclease digestion.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Scott Lujan

PROVIDER: E-GEOD-56939 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Heterogeneous polymerase fidelity and mismatch repair bias genome variation and composition.

Lujan Scott A SA Clausen Anders R AR Clark Alan B AB MacAlpine Heather K HK MacAlpine David M DM Malc Ewa P EP Mieczkowski Piotr A PA Burkholder Adam B AB Fargo David C DC Gordenin Dmitry A DA Kunkel Thomas A TA

Genome research 20140912 11

Mutational heterogeneity must be taken into account when reconstructing evolutionary histories, calibrating molecular clocks, and predicting links between genes and disease. Selective pressures and various DNA transactions have been invoked to explain the heterogeneous distribution of genetic variation between species, within populations, and in tissue-specific tumors. To examine relationships between such heterogeneity and variations in leading- and lagging-strand replication fidelity and misma ...[more]

PMID: 25217194

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Project description:DNA mismatch repair (MMR) is an evolutionarily conserved process that corrects innate DNA polymerase infidelities during replication to maintain genomic integrity. Defects in a subset of MMR genes are associated with hereditary non-polyposis colon cancer and some other sporadic cancers, highlighting the crucial role for MMR in genome maintenance. In E. coli a helicase implicated in the MMR process is well characterized, and named UvrD, whereas in mammals it has not been identified yet, even though the eukaryotic DNA mismatch pathway is analogous to the bacterial one and uses a similar repair mechanism and key components. Here we identify MCM9 as a helicase playing a vital role in MMR in mammals. MCM9 is the last discovered member of the MCM2-9 family, and has been implicated in replication and homologous recombination processes. By an affinity-purification proteomic approach, we found that MCM9 interacts with the MMR initiation complex. Immortalized cells from MCM9 knockout mice showed clear length alterations in their microsatellites, and a dramatic MMR deficiency compared to wild-type cells. We also found that a helicase-dead form of MCM9 is totally unable to restore the MMR deficiency phenotype. Furthermore, using an siRNA approach in human cells, we demonstrated that MCM9 is regulated by MSH2, a protein responsible for mismatch recognition. Our results clearly reveal that MCM9 functions as a helicase for DNA mismatch repair in mammals, and thus is essential for the maintenance of genome stability. Proteomics analysis: FLAG-HA-tagged human MCM9 plus associated proteins were isolated by tandem affinity purification from nuclear extracts of stably-expressing HeLa S3 cells, then analysed by SDS-PAGE and silver staining. Gel lanes were cut into slices, which were processed and analysed separately. Proteins in each gel slice were digested with trypsin, extracted and analysed by LC-MS/MS on a Thermo Scientific LTQ Velos mass spectrometer, generating a series of MS RAW files. Bioinformatics: Peptide and protein identification from MS data was performed using the Sequest program, with a human IPI sequence database (v.3.60). Trypsin was defined as the protease used, a peptide mass tolerance of 2.5 was specified, and all peptide matches have a Sequest Xcorr score ≥0.5.

Dataset Information

Interrelationships between replication infidelity and genome composition

Publications

Heterogeneous polymerase fidelity and mismatch repair bias genome variation and composition.

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