Genomic Instability Promoted by Overexpression of Mismatch Repair Factors in Yeast: A Model for Understanding Cancer Progression

Abstract

Mismatch repair (MMR) proteins act in spellchecker roles to excise misincorporation errors that occur during DNA replication. Curiously, large-scale analyses of a variety of cancers showed that increased expression of MMR proteins often correlated with tumor aggressiveness, metastasis, and early recurrence. To better understand these observations, we used The Cancer Genome Atlas and Gene Expression across Normal and Tumor tissue databases to analyze MMR protein expression in cancers. We found that the MMR genes and are overexpressed more frequently than , and that and are often cooverexpressed as a result of copy number amplifications of these genes. These observations encouraged us to test the effects of upregulating MMR protein levels in baker's yeast, where we can sensitively monitor genome instability phenotypes associated with cancer initiation and progression. Msh6 overexpression (two- to fourfold) almost completely disrupted mechanisms that prevent recombination between divergent DNA sequences by interacting with the DNA polymerase processivity clamp PCNA and by sequestering the Sgs1 helicase. Importantly, cooverexpression of Msh2 and Msh6 (∼eightfold) conferred, in a PCNA interaction-dependent manner, several genome instability phenotypes including increased mutation rate, increased sensitivity to the DNA replication inhibitor HU and the DNA-damaging agents MMS and 4-nitroquinoline N-oxide, and elevated loss-of-heterozygosity. Msh2 and Msh6 cooverexpression also altered the cell cycle distribution of exponentially growing cells, resulting in an increased fraction of unbudded cells, consistent with a larger percentage of cells in G1. These novel observations suggested that overexpression of MSH factors affected the integrity of the DNA replication fork, causing genome instability phenotypes that could be important for promoting cancer progression.

Overexpression of MutS impairs DNA mismatch repair and causes cell division defect inE.coli

“…We found two distinct phenotypes upon MutS-overexpression i.e., mutation phenotype and cell elongation phenotype. In yeast, overexpression of the primary mismatch repair complex (Msh2-Msh6) is shown to be mutagenic and the phenotype is attributed to replication fork instability due to enhanced Msh6-PCNA interaction 51 . In E.coli , if MutS-overexpression mediated mutations were due to replication fork instability, in our ssDNA recombineering experiments, MutS-overexpression should have equally rejected either G/t or C/a mismatch generating ssDNA oligo.…”

Chromatin Modifiers Alter Recombination Between Divergent DNA Sequences

“…Previously, we showed that cooverexpression of the Msh2 and Msh6 MMR proteins in wild-type strains increased the frequency of heteroduplex rejection in the inverted repeat assay by 3.5-fold, and hypothesized that this was due to the increased availability of functional MSH complexes acting in heteroduplex rejection (Chakraborty et al , 2018. Because both the sir2D mutation, and Msh2 and Msh6 cooverexpression increased rejection, we asked if the increased rejection seen in sir2D strains would rise to an even higher level in the presence of pEAM272 (2m, MSH2, MSH6 plasmid).…”

“…The mutations comprising the pol30-8 allele (R61A, D63A) were introduced into pEAA578 (POL30::KANMX) by Q5 site-directed mutagenesis (New England Biolabs, Beverly, MA) to create the single-step integrating vector pEAA633 (pol30-8::KANMX). Plasmids pRS426 (2m, URA3) (Christianson et al 1992) and pEAM272 (MSH2, MSH6, 2m, URA3) ( Chakraborty et al 2018) were used in the MSH overexpression experiments presented in Table 2.…”

Chromatin modifiers alter recombination between divergent DNA sequences

“…Such an experiment tests if the increases in rejection seen in the two conditions reflect common or distinct regulatory steps. In wild-type strains bearing pEAM272 the Msh2-Msh6 complex is overexpressed by ~8-fold (Chakraborty, Dinh and Alani 2018). Wild-type strains containing pEAM272 display a 4.3-fold increase, compared to those containing the empty vector pRS426, in the rejection ratio, consistent with an increased concentration of Msh2-Msh6 resulting in improved rejection by increasing the likelihood of mismatch recognition in heteroduplex DNA (Table 2).…”