RESUMO
The DNA mismatch repair (MMR) system plays a crucial role in the prevention of replication errors and in the correction of some oxidative damages of DNA bases. In the present work the most abundant oxidized pyrimidine lesion, 5,6-dihydro-5,6-dihydroxythymidine (thymidine glycol, Tg) was tested for being recognized and processed by the E. coli MMR system, namely complex of MutS, MutL and MutH proteins. In a partially reconstituted MMR system with MutS-MutL-MutH proteins, G/Tg and A/Tg containing plasmids failed to provoke the incision of DNA. Tg residue in the 30-mer DNA duplex destabilized double helix due to stacking disruption with neighboring bases. However, such local structural changes are not important for E. coli MMR system to recognize this lesion. A lack of repair of Tg containing DNA could be due to a failure of MutS (a first acting protein of MMR system) to interact with modified DNA in a proper way. It was shown that Tg in DNA does not affect on ATPase activity of MutS. On the other hand, MutS binding affinities to DNA containing Tg in G/Tg and A/Tg pairs are lower than to DNA with a G/T mismatch and similar to canonical DNA. Peculiarities of MutS interaction with DNA was monitored by Förster resonance energy transfer (FRET) and fluorescence anisotropy. Binding of MutS to Tg containing DNAs did not result in the formation of characteristic DNA kink. Nevertheless, MutS homodimer orientation on Tg-DNA is similar to that in the case of G/T-DNA. In contrast to G/T-DNA, neither G/Tg- nor A/Tg-DNA was able to stimulate ADP release from MutS better than canonical DNA. Thus, Tg residue in DNA is unlikely to be recognized or processed by the E. coli MMR system. Probably, the MutS transformation to active "sliding clamp" conformation on Tg-DNA is problematic.
Assuntos
Reparo de Erro de Pareamento de DNA , DNA Bacteriano/genética , Escherichia coli/genética , Timidina/análogos & derivados , Adenosina Trifosfatases/química , Adenosina Trifosfatases/fisiologia , Trifosfato de Adenosina/química , Clivagem do DNA , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/fisiologia , DNA Bacteriano/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/fisiologia , Endodesoxirribonucleases/química , Endodesoxirribonucleases/fisiologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/fisiologia , Hidrólise , Proteínas MutL , Proteína MutS de Ligação de DNA com Erro de Pareamento/química , Proteína MutS de Ligação de DNA com Erro de Pareamento/fisiologia , Plasmídeos/química , Plasmídeos/genética , Ligação Proteica , Timidina/química , Timidina/genéticaRESUMO
The DNA repair protein MutS forms clamp-like structures on DNA that search for and recognize base mismatches leading to ATP-transformed signaling clamps. In this study, the mobile MutS clamps were trapped on DNA in a functional state using single-cysteine variants of MutS and thiol-modified homoduplex or heteroduplex DNA. This approach allows stabilization of various transient MutS-DNA complexes and will enable their structural and functional analysis.
Assuntos
Reparo de Erro de Pareamento de DNA , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Pareamento Incorreto de Bases , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteína MutS de Ligação de DNA com Erro de Pareamento/química , Ácidos Nucleicos Heteroduplexes/genética , Ácidos Nucleicos Heteroduplexes/metabolismo , Transdução de SinaisRESUMO
The ternary complex comprising MutS, MutL, and DNA is a key intermediate in DNA mismatch repair. We used chemical cross-linking and fluorescence resonance energy transfer (FRET) to study the interaction between MutS and MutL and to shed light onto the structure of this complex. Via chemical cross-linking, we could stabilize this dynamic complex and identify the structural features of key events in DNA mismatch repair. We could show that in the complex between MutS and MutL the mismatch-binding and connector domains of MutS are in proximity to the N-terminal ATPase domain of MutL. The DNA- and nucleotide-dependent complex formation could be monitored by FRET using single cysteine variants labeled in the connector domain of MutS and the transducer domain of MutL, respectively. In addition, we could trap MutS after an ATP-induced conformational change by an intramolecular cross-link between Cys-93 of the mismatch-binding domain and Cys-239 of the connector domain.
Assuntos
Adenosina Trifosfatases/química , Reparo de Erro de Pareamento de DNA , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Regulação Bacteriana da Expressão Gênica , Proteína MutS de Ligação de DNA com Erro de Pareamento/química , Trifosfato de Adenosina/química , Reagentes de Ligações Cruzadas/química , Reagentes de Ligações Cruzadas/farmacologia , Cisteína/química , Reparo do DNA , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Proteínas de Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas MutL , Mutagênese Sítio-Dirigida , Conformação Proteica , Estrutura Terciária de Proteína , UltracentrifugaçãoRESUMO
DNA mismatch repair (MMR) and very-short patch (VSP) repair are two pathways involved in the repair of T:G mismatches. To learn about competition and cooperation between these two repair pathways, we analyzed the physical and functional interaction between MutL and Vsr using biophysical and biochemical methods. Analytical ultracentrifugation reveals a nucleotide-dependent interaction between Vsr and the N-terminal domain of MutL. Using chemical crosslinking, we mapped the interaction site of MutL for Vsr to a region between the N-terminal domains similar to that described before for the interaction between MutL and the strand discrimination endonuclease MutH of the MMR system. Competition between MutH and Vsr for binding to MutL resulted in inhibition of the mismatch-provoked MutS- and MutL-dependent activation of MutH, which explains the mutagenic effect of Vsr overexpression. Cooperation between MMR and VSP repair was demonstrated by the stimulation of the Vsr endonuclease in a MutS-, MutL- and ATP-hydrolysis-dependent manner, in agreement with the enhancement of VSP repair by MutS and MutL in vivo. These data suggest a mobile MutS-MutL complex in MMR signalling, that leaves the DNA mismatch prior to, or at the time of, activation of downstream effector molecules such as Vsr or MutH.