RESUMO
Mutations in the human mismatch repair (MMR) gene hMSH2 have been linked to approximately 40% of hereditary nonpolyposis colorectal cancers (HNPCC). While the consequences of deletion or truncating mutations of hMSH2 would appear clear, the detailed functional defects associated with missense alterations are unknown. We have examined the effect of seven single amino acid substitutions associated with HNPCC that cover the structural subdomains of the hMSH2 protein. We show that alterations which produced a known cancer-causing phenotype affected the mismatch-dependent molecular switch function of the biologically relevant hMSH2-hMSH6 heterodimer. Our observations demonstrate that amino acid substitutions within hMSH2 that are distant from known functional regions significantly alter biochemical activity and the ability of hMSH2-hMSH6 to form a sliding clamp.
Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Proteínas de Ligação a DNA/genética , Proteínas Proto-Oncogênicas/genética , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Pareamento Incorreto de Bases/genética , Sítios de Ligação/genética , DNA/metabolismo , Reparo do DNA/genética , Dimerização , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Hidrólise , Proteína 2 Homóloga a MutS , Mutagênese/genética , Mutação de Sentido Incorreto/genética , Ligação Proteica/genéticaRESUMO
We report that Plasmodium falciparum (Pf) encodes a 912 amino acid ATP-dependent DNA ligase. Protein sequence analysis of Pf DNA ligase I indicates a strong sequence similarity, particularly in the C-terminal region, to DNA ligase I homologues. The activity of recombinant Pf DNA ligase I (PfLigI) was investigated using protein expressed in HEK293 cells. The PfLigI gene product is approximately 94kDa and catalyzes phosphodiester bond formation on a singly nicked DNA substrate. The enzyme is most active at alkaline pH (8.5) and with Mg(2+) or Mn(2+) and ATP as cofactors. Kinetic studies of PfLigI revealed that the enzyme has similar substrate affinity (K(m) 2.6nM) as compared to human DNA ligase I and k(cat) (2.3x10(-3)s(-1)) and k(cat)/K(m) (8.8x10(5)M(-1)s(-1)) which are similar to other ATP-dependent DNA ligases. PfLigI was able to join RNA-DNA substrates only when the RNA sequence was upstream of the nick, confirming that it is DNA ligase I and has no associated DNA ligase III like activity.
Assuntos
DNA Ligases/genética , DNA Ligases/metabolismo , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Cátions Bivalentes/metabolismo , Linhagem Celular , Clonagem Molecular , Coenzimas/metabolismo , DNA/metabolismo , DNA Ligase Dependente de ATP , DNA Ligases/química , Estabilidade Enzimática , Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Magnésio/metabolismo , Manganês/metabolismo , Dados de Sequência Molecular , Plasmodium falciparum/genética , Proteínas de Protozoários/química , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Malarial parasites exhibit striking genetic plasticity, a hallmark of which is an ever-increasing rate of resistance to new drugs, especially in Southeast Asia where multi-drug resistance (MDR) threatens the last line of antimalarial drugs, the artesunate compounds. Previous studies quantified the accelerated resistance to multiple drugs (ARMD) phenomenon, but the underpinning mechanism(s) remains unknown. We utilize a forward genetic assay to investigate a new hypothesis that defective DNA mismatch repair (MMR) contributes to the development of MDR by Plasmodium falciparum parasites. We report that two ARMD parasites, W2 and Dd2, have defective MMR, as do the chloroquine-resistant parasites T9-94, 7C12, and 7G8. By contrast, the chloroquine-sensitive parasites HB3, D6 and 3D7 were MMR proficient. Interestingly, W2 was unable to repair substrates with a strand break located 3' to the mismatch, which is attributable to a large observed decrease in PfMutLα content. These data imply that antimalarial drug resistance can result from defective MMR.
Assuntos
Antimaláricos/farmacologia , Reparo de Erro de Pareamento de DNA , Distúrbios no Reparo do DNA , Resistência a Medicamentos , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Plasmodium falciparum/genéticaRESUMO
Five MutS homologs (MSH), which form three heterodimeric protein complexes, have been identified in eukaryotes. While the human hMSH2-hMSH3 and hMSH2-hMSH6 heterodimers operate primarily in mitotic mismatch repair (MMR), the biochemical function(s) of the meiosis-specific hMSH4-hMSH5 heterodimer is unknown. Here, we demonstrate that purified hMSH4-hMSH5 binds uniquely to Holliday Junctions. Holliday Junctions stimulate the hMSH4-hMSH5 ATP hydrolysis (ATPase) activity, which is controlled by Holliday Junction-provoked ADP-->ATP exchange. ATP binding by hMSH4-hMSH5 induces the formation of a hydrolysis-independent sliding clamp that dissociates from the Holliday Junction crossover region, embracing two homologous duplex DNA arms. Fundamental differences between hMSH2-hMSH6 and hMSH4-hMSH5 Holliday Junction recognition are detailed. Our results support the attractive possibility that hMSH4-hMSH5 stabilizes and preserves a meiotic bimolecular double-strand break repair (DSBR) intermediate.