Both microsatellite length and sequence context determine frameshift mutation rates in defective DNA mismatch repair

Abstract

It is generally accepted that longer microsatellites mutate more frequently in defective DNA mismatch repair (MMR) than shorter microsatellites. Indeed, we have previously observed that the A10 microsatellite of transforming growth factor beta type II receptor (TGFBR2) frameshifts -1 bp at a faster rate than the A8 microsatellite of activin type II receptor (ACVR2), although both genes become frameshift-mutated in >80% of MMR-defective colorectal cancers. To experimentally determine the effect of microsatellite length upon frameshift mutation in gene-specific sequence contexts, we altered the microsatellite length within TGFBR2 exon 3 and ACVR2 exon 10, generating A7, A10 and A13 constructs. These constructs were cloned 1 bp out of frame of EGFP, allowing a -1 bp frameshift to drive EGFP expression, and stably transfected into MMR-deficient cells. Subsequent non-fluorescent cells were sorted, cultured for 7-35 days and harvested for EGFP analysis and DNA sequencing. Longer microsatellites within TGFBR2 and ACVR2 showed significantly higher mutation rates than shorter ones, with TGFBR2 A13, A10 and A7 frameshifts measured at 22.38x10(-4), 2.17x10(-4) and 0.13x10(-4), respectively. Surprisingly, shorter ACVR2 constructs showed three times higher mutation rates at A7 and A10 lengths than identical length TGFBR2 constructs but comparably lower at the A13 length, suggesting influences from both microsatellite length as well as the sequence context. Furthermore, the TGFBR2 A13 construct mutated into 33% A11 sequences (-2 bp) in addition to expected A12 (-1 bp), indicating that this construct undergoes continual subsequent frameshift mutation. These data demonstrate experimentally that both the length of a mononucleotide microsatellite and its sequence context influence mutation rate in defective DNA MMR.

A Polymerase – Tautomeric Model for Targeted Frameshift Mutations: Deletions Formation during Error-prone or SOS Replication of Double-stranded DNA Containing cis-syn Cyclobutane Thymine Dimers

“…Frequencies of frameshift mutations increase as a function of the number of reiterated base pairs at DNA sites [27]. Both the length of a mononucleotide microsatellite and its sequence context influence mutation rate in defective DNA in Error-prone or SOS Replication of Double-stranded DNA Containing cis-syn Cyclobutane Thymine Dimers mismatch repair (MMR)-deficient cells [28]. Ultraviolet radiation C (UVC) or reactive oxygen species-induced CC to TT tandem mutations is markedly enhanced in MMRdeficient cells.…”

Microsatellite instability in colorectal cancer: from molecular oncogenic mechanisms to clinical implications

“…In absence of functional studies, discriminating between the genes that are true targets for colorectal MSI-driven mutagenesis from those whose mutations occur randomly and are left unrepaired due to the absence of MMR is not self-evident. High mutation prevalence in a given gene is generally regarded as strongly indicating its oncogenic role, although mutation rates of microsatellites are extremely variable, being influenced by flanking sequences, sequence composition, motif unit size, and recombination rate [26,36,37]. A bi-allelic inactivation and a role in a growth suppressor pathway are among other widely accepted criteria, but no consensus has been achieved, especially no mutation incidence cut-off value could be defined [18,48].…”

973 Relationship of EMAST and Microsatellite Instability Among Patients With Rectal Cancer

“…Yamada et al have proposed a panel of 10 tetranucleotide in addition to the five loci tested in our study, including L17835, D19S394, L17686, UT5320, and D11S488. 23 Because of the polymorphic nature of tetranucleotide repeats and the ability of microsatellites to have varying mutation rates based on their sequence context, 24 we used two or more positive markers to define EMAST. In addition, the majority of the limited studies on EMAST have used two or more tetranucleotide loci instability as criteria for determination of EMAST.…”