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1.
Int J Mol Sci ; 15(8): 13932-7, 2014 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-25116689

RESUMO

Mutations in human DNA mismatch repair (MMR) genes are commonly associated with hereditary nonpolyposis colorectal cancer (HNPCC). MLH1 protein heterodimerizes with PMS2, PMS1, and MLH3 to form MutLα, MutLß, and MutLγ, respectively. We reported recently stable expression of GFP-linked MLH3 in human cell lines. Monitoring these cell lines during the cell cycle using live cell imaging combined with confocal microscopy, we detected accumulation of MLH3 at the centrosomes. Fluorescence recovery after photobleaching (FRAP) revealed high mobility and fast exchange rates at the centrosomes as it has been reported for other DNA repair proteins. MLH3 may have a role in combination with other repair proteins in the control of centrosome numbers.


Assuntos
Proteínas de Transporte/metabolismo , Centrossomo/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Células HEK293 , Humanos , Microscopia Confocal , Proteínas MutL
2.
J Cell Biochem ; 114(10): 2405-14, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23696135

RESUMO

The human DNA mismatch repair (MMR) gene family comprises four MutL paralogues capable of forming heterodimeric MutLα (MLH1-PMS2), MutLß (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.


Assuntos
Reparo de Erro de Pareamento de DNA/fisiologia , Enzimas Reparadoras do DNA/metabolismo , DNA/genética , Ciclo Celular/genética , Ciclo Celular/fisiologia , Linhagem Celular , Ensaio Cometa , DNA/metabolismo , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo de Erro de Pareamento de DNA/genética , Enzimas Reparadoras do DNA/genética , Humanos , Imunoprecipitação
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