Stable expression of MutLγ in human cells reveals no specific response to mismatched DNA, but distinct recruitment to damage sites.

2.50
Hdl Handle:
http://hdl.handle.net/10033/305659
Title:
Stable expression of MutLγ in human cells reveals no specific response to mismatched DNA, but distinct recruitment to damage sites.
Authors:
Roesner, Lennart M; Mielke, Christian; Fähnrich, Silke; Merkhoffer, Yvonne; Dittmar, Kurt E J; Drexler, Hans G; Dirks, Wilhelm G
Abstract:
The human DNA mismatch repair (MMR) gene family comprises four MutL paralogues capable of forming heterodimeric MutLα (MLH1-PMS2), MutLβ (MLH1-PMS1), and MutLγ (MLH1-MLH3) protein complexes. Human MutL subunits PMS2 and MLH3 contain an evolutionarily conserved amino acid motif DQHA(X)2E(X)4E identified as an endonucleolytic domain capable of incising a defective DNA strand. PMS2 of MutLα is generally accepted to be the sole executor of endonucleolytic activity, but since MLH3 was shown to be able to perform DNA repair at low levels in vitro, our aim was to investigate whether or not MLH3 is activated as a backup under MutLα-deficient conditions. Here, we report stable expression of GFP-tagged MLH3 in the isogenic cell lines 293 and 293T which are functional or defective for MLH1 expression, respectively. As expected, MLH3 formed dimeric complexes with endogenous and recombinant MLH1. MutLγ dimers were recruited to sites of DNA damage induced by UVA micro-irradiation as shown for MutLα. Surprisingly, splicing variant MLH3Δ7 lacking the endonucleolytic motif displayed congruent foci formation, implying that recruitment is not necessarily representing active DNA repair. As an alternative test for repair enzyme activity, we combined alkylation-directed DNA damage with comet formation assays. While recombinant MutLα led to full recovery of DNA damage response in MMR deficient cells, expression of MutLγ or single MLH3 failed to do so. These experiments show recruitment and persistence of MutLγ-heterodimers at UVA-induced DNA lesions. However, we demonstrate that in a MutLα-deficient background no DNA repair-specific function carried out by MutLγ can be detected in living cells.
Affiliation:
Department of Human and Animal Cell Lines, Leibniz-Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany. roesner.lennart@mh-hannover.de
Citation:
Stable expression of MutLγ in human cells reveals no specific response to mismatched DNA, but distinct recruitment to damage sites. 2013, 114 (10):2405-14 J. Cell. Biochem.
Journal:
Journal of cellular biochemistry
Issue Date:
Oct-2013
URI:
http://hdl.handle.net/10033/305659
DOI:
10.1002/jcb.24591
PubMed ID:
23696135
Type:
Article
Language:
en
ISSN:
1097-4644
Appears in Collections:
Publications of Dept. Gene Regulation and Differentiation (RDIF)

Full metadata record

DC FieldValue Language
dc.contributor.authorRoesner, Lennart Men
dc.contributor.authorMielke, Christianen
dc.contributor.authorFähnrich, Silkeen
dc.contributor.authorMerkhoffer, Yvonneen
dc.contributor.authorDittmar, Kurt E Jen
dc.contributor.authorDrexler, Hans Gen
dc.contributor.authorDirks, Wilhelm Gen
dc.date.accessioned2013-11-21T11:45:20Z-
dc.date.available2013-11-21T11:45:20Z-
dc.date.issued2013-10-
dc.identifier.citationStable expression of MutLγ in human cells reveals no specific response to mismatched DNA, but distinct recruitment to damage sites. 2013, 114 (10):2405-14 J. Cell. Biochem.en
dc.identifier.issn1097-4644-
dc.identifier.pmid23696135-
dc.identifier.doi10.1002/jcb.24591-
dc.identifier.urihttp://hdl.handle.net/10033/305659-
dc.description.abstractThe human DNA mismatch repair (MMR) gene family comprises four MutL paralogues capable of forming heterodimeric MutLα (MLH1-PMS2), MutLβ (MLH1-PMS1), and MutLγ (MLH1-MLH3) protein complexes. Human MutL subunits PMS2 and MLH3 contain an evolutionarily conserved amino acid motif DQHA(X)2E(X)4E identified as an endonucleolytic domain capable of incising a defective DNA strand. PMS2 of MutLα is generally accepted to be the sole executor of endonucleolytic activity, but since MLH3 was shown to be able to perform DNA repair at low levels in vitro, our aim was to investigate whether or not MLH3 is activated as a backup under MutLα-deficient conditions. Here, we report stable expression of GFP-tagged MLH3 in the isogenic cell lines 293 and 293T which are functional or defective for MLH1 expression, respectively. As expected, MLH3 formed dimeric complexes with endogenous and recombinant MLH1. MutLγ dimers were recruited to sites of DNA damage induced by UVA micro-irradiation as shown for MutLα. Surprisingly, splicing variant MLH3Δ7 lacking the endonucleolytic motif displayed congruent foci formation, implying that recruitment is not necessarily representing active DNA repair. As an alternative test for repair enzyme activity, we combined alkylation-directed DNA damage with comet formation assays. While recombinant MutLα led to full recovery of DNA damage response in MMR deficient cells, expression of MutLγ or single MLH3 failed to do so. These experiments show recruitment and persistence of MutLγ-heterodimers at UVA-induced DNA lesions. However, we demonstrate that in a MutLα-deficient background no DNA repair-specific function carried out by MutLγ can be detected in living cells.en
dc.language.isoenen
dc.rightsArchived with thanks to Journal of cellular biochemistryen
dc.titleStable expression of MutLγ in human cells reveals no specific response to mismatched DNA, but distinct recruitment to damage sites.en
dc.typeArticleen
dc.contributor.departmentDepartment of Human and Animal Cell Lines, Leibniz-Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany. roesner.lennart@mh-hannover.deen
dc.identifier.journalJournal of cellular biochemistryen

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