ABSTRACT
Mismatch repair (MMR) is activated by evolutionarily conserved MutS homologs (MSH) and MutL homologs (MLH/PMS). MSH recognizes mismatched nucleotides and form extremely stable sliding clamps that may be bound by MLH/PMS to ultimately authorize strand-specific excision starting at a distant 3'- or 5'-DNA scission. The mechanical processes associated with a complete MMR reaction remain enigmatic. The purified human (Homo sapien or Hs) 5'-MMR excision reaction requires the HsMSH2-HsMSH6 heterodimer, the 5' â 3' exonuclease HsEXOI, and the single-stranded binding heterotrimer HsRPA. The HsMLH1-HsPMS2 heterodimer substantially influences 5'-MMR excision in cell extracts but is not required in the purified system. Using real-time single-molecule imaging, we show that HsRPA or Escherichia coli EcSSB restricts HsEXOI excision activity on nicked or gapped DNA. HsMSH2-HsMSH6 activates HsEXOI by overcoming HsRPA/EcSSB inhibition and exploits multiple dynamic sliding clamps to increase tract length. Conversely, HsMLH1-HsPMS2 regulates tract length by controlling the number of excision complexes, providing a link to 5' MMR.
Subject(s)
Base Pair Mismatch , DNA Repair , Adaptor Proteins, Signal Transducing/metabolism , Adenosine Triphosphatases/metabolism , DNA Repair Enzymes/metabolism , DNA-Binding Proteins/metabolism , Dimerization , Humans , Mismatch Repair Endonuclease PMS2 , MutL Protein Homolog 1 , Nuclear Proteins/metabolismABSTRACT
High fidelity homologous DNA recombination depends on mismatch repair (MMR), which antagonizes recombination between divergent sequences by rejecting heteroduplex DNA containing excessive nucleotide mismatches. The hMSH2-hMSH6 heterodimer is the first responder in postreplicative MMR and also plays a prominent role in heteroduplex rejection. Whether a similar molecular mechanism underlies its function in these two processes remains enigmatic. We have determined that hMSH2-hMSH6 efficiently recognizes mismatches within a D-loop recombination initiation intermediate. Mismatch recognition by hMSH2-hMSH6 is not abrogated by human replication protein A (HsRPA) bound to the displaced single-stranded DNA (ssDNA) or by HsRAD51. In addition, ATP-bound hMSH2-hMSH6 sliding clamps that are essential for downstream MMR processes are formed and constrained within the heteroduplex region of the D-loop. Moreover, the hMSH2-hMSH6 sliding clamps are stabilized on the D-loop by HsRPA bound to the displaced ssDNA. Our findings reveal similarities and differences in hMSH2-hMSH6 mismatch recognition and sliding-clamp formation between a D-loop recombination intermediate and linear duplex DNA.
Subject(s)
DNA Mismatch Repair , DNA-Binding Proteins/chemistry , DNA/chemistry , MutS Homolog 2 Protein/chemistry , Recombination, Genetic , Adenosine Diphosphate/chemistry , Adenosine Triphosphate/chemistry , Base Pair Mismatch , Biotinylation , Humans , Hydrolysis , Kinetics , Protein Binding , Protein Structure, Tertiary , Rad51 Recombinase/chemistry , Replication Protein A/chemistryABSTRACT
The majority of Lynch syndrome (LS), also known as hereditary non-polyposis colorectal cancer (HNPCC), has been linked to heterozygous defects in DNA mismatch repair (MMR). MMR is a highly conserved pathway that recognizes and repairs polymerase misincorporation errors and nucleotide damage as well as functioning as a damage sensor that signals apoptosis. Loss-of-heterozygosity (LOH) that retains the mutant MMR allele and epigenetic silencing of MMR genes are associated with an increased mutation rate that drives carcinogenesis as well as microsatellite instability that is a hallmark of LS/HNPCC. Understanding the biophysical functions of the MMR components is crucial to elucidating the role of MMR in human tumorigenesis and determining the pathogenetic consequences of patients that present in the clinic with an uncharacterized variant of the MMR genes. We summarize the historical association between LS/HNPCC and MMR, discuss the mechanism of the MMR and finally examine the functional analysis of MMR defects found in LS/HNPCC patients and their relationship with the severity of the disease.
Subject(s)
Colorectal Neoplasms, Hereditary Nonpolyposis/genetics , DNA Mismatch Repair/genetics , HumansABSTRACT
The hMSH2(M688R) mismatch repair (MMR) gene mutation has been found in five large families from Tenerife, Spain, suggesting it is a Lynch syndrome or hereditary non-polyposis colorectal cancer (LS/HNPCC) founder mutation. In addition to classical LS/HNPCC tumors, these families present with a high incidence of central nervous system (CNS) tumors normally associated with Turcot or constitutional mismatch repair deficiency (CMMR-D) syndromes. Turcot and CMMR-D mutations may be biallelic, knocking out both copies of the MMR gene. The hMSH2(M688R) mutation is located in the ATP hydrolysis (ATPase) domain. We show that the hMSH2(M688R)-hMSH6 heterodimer binds to mismatched nucleotides but lacks normal ATP functions and inhibits MMR in vitro when mixed with the wild-type (WT) heterodimer. Another alteration that has been associated with LS/HNPCC, hMSH2(M688I)-hMSH6, displays no identifiable differences with the WT heterodimer. Interestingly, some extracolonic tumors from hMSH2(M688R) carriers may express hMSH2-hMSH6, yet display microsatellite instability (MSI). The functional analysis along with variability in tumor expression and the high incidence of CNS tumors suggests that hMSH2(M688R) may act as a dominant negative in some tissues, while the hMSH2(M688I) is most likely a benign polymorphism.
Subject(s)
Colorectal Neoplasms, Hereditary Nonpolyposis/genetics , MutS Homolog 2 Protein/genetics , Mutation , Amino Acid Sequence , DNA Mismatch Repair , DNA-Binding Proteins/genetics , Humans , Immunohistochemistry , Molecular Sequence Data , MutS Homolog 2 Protein/analysis , MutS Homolog 2 Protein/physiologyABSTRACT
Recent data show that more than 50% of catheter-associated bloodstream infections are caused by staphylococci. Staphylococcal infections produced by intercellular-adhesion cluster (ica) carriers can be even more problematic due to the presence of methicillin and mupirocin resistance genes. In the present study, a multiplex PCR protocol that allows the simultaneous identification of staphylococci and detection of both the ica and methicillin and/or mupirocin resistance genes was developed. Furthermore, the method allows differential detection of the ica locus from Staphylococcus aureus and Staphylococcus epidermidis.
Subject(s)
Catheters, Indwelling , Drug Resistance, Bacterial/genetics , Methicillin Resistance/genetics , Mupirocin/pharmacology , Staphylococcus/genetics , Base Sequence , DNA Primers , Humans , Polymerase Chain Reaction/methods , Staphylococcus/drug effects , Staphylococcus/isolation & purification , Staphylococcus aureus/drug effects , Staphylococcus aureus/genetics , Staphylococcus aureus/isolation & purification , Staphylococcus epidermidis/drug effects , Staphylococcus epidermidis/genetics , Staphylococcus epidermidis/isolation & purificationABSTRACT
Recent data show that more than 50% of catheter-associated bloodstream infections are caused by staphylococci. Staphylococcal infections produced by intercellular-adhesion cluster (ica) carriers can be even more problematic due to the presence of methicillin and mupirocin resistance genes. In the present study, a multiplex PCR protocol that allows the simultaneous identification of staphylococci and detection of both the ica and methicillin and/or mupirocin resistance genes was developed. Furthermore, the method allows differential detection of the ica locus from Staphylococcus aureus and Staphylococcus epidermidis (AU)
Estudios recientes han demostrado que más del 50 por ciento de las septicemias asociadas al uso de catéteres están causadas por estafilococos. Las infecciones estafilococales producidas por cepas portadoras del operón de adhesión intercelular (ica) pueden agravarse por la presencia de genes de resistencia a la meticilina y/o a la mupirocina. Hemos desarrollado un protocolo de PCR múltiple que permite, simultáneamente, identificar estafilococos y detectar la presencia de ica y de los genes de resistencia a la meticilina y/o a la mupirocina. Además, dicho método permite la detección diferencial del locus ica de Staphylococcus aureus y/o S. epidermidis (AU)
