ABSTRACT
Exonucleolytic degradation of DNA is an essential part of many DNA metabolic processes including DNA mismatch repair (MMR) and recombination. Human exonuclease I (hExoI) is a member of a family of conserved 5' --> 3' exonucleases, which are implicated in these processes by genetic studies. Here, we demonstrate that hExoI binds strongly to hMLH1, and we describe interaction regions between hExoI and the MMR proteins hMSH2, hMSH3, and hMLH1. In addition, hExoI forms an immunoprecipitable complex with hMLH1/hPMS2 in vivo. The study of interaction regions suggests a biochemical mechanism of the involvement of hExoI as a downstream effector in MMR and/or DNA recombination.
Subject(s)
Base Pair Mismatch , DNA Repair , DNA-Binding Proteins/metabolism , Exodeoxyribonucleases/metabolism , Multidrug Resistance-Associated Proteins , Neoplasm Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Adaptor Proteins, Signal Transducing , Binding Sites , Carrier Proteins , Cloning, Molecular , DNA Repair Enzymes , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Exodeoxyribonucleases/chemistry , Exodeoxyribonucleases/genetics , Humans , MutL Protein Homolog 1 , MutS Homolog 2 Protein , MutS Homolog 3 Protein , Neoplasm Proteins/chemistry , Neoplasm Proteins/genetics , Nuclear Proteins , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Sequence Deletion , Substrate SpecificityABSTRACT
Human Rad51 (hRad51) has been found to be associated with BRCA1, BRCA2, and p53 either directly or indirectly and is one of at least eight human genes that are members of the Escherichia coli RecA/Saccharomyces cerevisiae Rad51 family thought to affect genomic stability through DNA recombination/repair processes. While inactivation of DNA mismatch repair clearly leads to instability of repeated sequences and to an increased risk for tumorigenesis, such a parallel for the RecA family members has not been reported. Recently, a high frequency of loss of heterozygosity at chromosome 15q14-15, near the genomic region containing hRad51, has been reported in human tumors (W. Wick et al., Oncogene, 12: 973-978, 1996). To determine whether hRad51 inactivation may be involved in the etiology of these tumors, we have characterized the hRad51 genetic locus and mapped it to chromosome 15q14-15 within the central region of loss of heterozygosity. However, single-strand conformational polymorphism analysis and direct sequencing of tumors did not reveal any mutations in the hRad51 coding sequence or intron/exon boundaries. We also examined the DNA methylation status of a CpG-rich region in the putative hRad51 promoter region. No indication of hypermethylation was found. These results suggest that hRad51 is not a tumor suppressor because it is either an essential gene, redundant gene and/or independent of the BRCA1/BRCA2 tumor suppressor pathway(s).
Subject(s)
Chromosomes, Human, Pair 15 , DNA-Binding Proteins/genetics , Loss of Heterozygosity , Neoplasms/genetics , Polymorphism, Single-Stranded Conformational , 5' Untranslated Regions/genetics , Base Sequence , Chromosome Mapping , DNA Methylation , Dinucleoside Phosphates/analysis , Exons , Humans , Introns , Molecular Sequence Data , Rad51 Recombinase , Rec A Recombinases/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae ProteinsABSTRACT
DNA mismatch repair (MMR) plays a vital role in the faithful replication of DNA, and its inactivation leads to a mutator phenotype that has been associated with the common cancer susceptibility syndrome Hereditary Non-Polyposis Colorectal Cancer (HNPCC). Here, we report on a novel human exonuclease (hExoI) that is related to the yeast exonuclease 1. The hExoI cDNA comprises 2541 bp, which code for a Mr 94,000 protein that appears to be highly expressed in testis tissue and at very low levels in other tissues. The hExoI gene has 14 exons and is located on chromosome 1q43, as determined by fluorescence in situ hybridization and radiation hybrid mapping. hExoI was found to interact strongly with the human MMR protein hMSH2, suggesting its involvement in the MMR process and/or DNA recombination.
Subject(s)
DNA Repair , DNA-Binding Proteins , Exodeoxyribonucleases/metabolism , Proto-Oncogene Proteins/metabolism , Amino Acid Sequence , Base Sequence , Chromosome Mapping , DNA Repair Enzymes , Exodeoxyribonucleases/genetics , Humans , Molecular Sequence Data , MutS Homolog 2 Protein , Polymerase Chain ReactionABSTRACT
Astronauts, particularly in Skylab flights, experienced varying degrees of motion sickness lasting 3-5 d. One possible mechanism for this motion sickness adaptation is believed to be a reduction in otolith sensitivity with an attendant reduction in sensory conflict. In an attempt to determine if this hypothesis is valid, a ground-based pilot study was conducted on a vertical linear accelerator. The extent of habituation to accelerations which initially produced motion sickness was evaluated, along with the possible value of habituation training to minimize the space motion sickness problem. Results showed that habituation occurred for 6 of the 8 subjects tested. However, in tests designed to measure dynamic and static otolith function, no significant differences between pre- and post-habituation tests were observed. Cross habituation effects to a standard Coriolis acceleration test were not significant. It is unlikely that ground-based pre-habituation to linear accelerations of the type examined would alter susceptibility to space motion sickness.
