C-terminal fluorescent labeling impairs functionality of DNA mismatch repair proteins.

PloS One
Angela BriegerStefan Zeuzem

Abstract

The human DNA mismatch repair (MMR) process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2). Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-termi...Continue Reading

References

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Citations

Jul 25, 2012·Hereditary Cancer in Clinical Practice·Christopher D Heinen, Lene Juel Rasmussen
Mar 22, 2013·Bioconjugate Chemistry·Pengcheng ZhangHonggang Cui
Dec 29, 2015·DNA Repair·Javier Peña-Diaz, Lene Juel Rasmussen
Jan 5, 2016·DNA Repair·Tobias T Schmidt, Hans Hombauer
Jan 13, 2016·DNA Repair·Jared M FischerR Michael Liskay
Jun 28, 2012·Human Mutation·Ester BorràsGabriel Capellá
Dec 5, 2014·Organic & Biomolecular Chemistry·Marta SolerLidia Feliu
Sep 12, 2017·Chemical Communications : Chem Comm·Christoph NitscheGottfried Otting
May 28, 2020·Open Biology·Navaneethan PalanisamyBarbara Di Ventura
Dec 19, 2020·Journal of Biomolecular Structure & Dynamics·Mohammad KhedriNima Rezaei
Aug 28, 2021·International Journal of Molecular Sciences·Jane H FrederiksenThomas V O Hansen

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Methods Mentioned

BETA
transfection
FCS
confocal laser microscopy

Software Mentioned

Rad Quantity One
Multi Gauge
Quantity One
Bio
PyMol

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