Biochemical studies of homologous and nonhomologous recombination in human cells.
Purified and partially purified protein fractions from human cells have been developed that promote homologous and nonhomologous recombination reactions in vitro. Homologous pairing of model DNA substrates is catalyzed by the homologous pairing protein HPP-1 in a magnesium-dependent, ATP-independent reaction that requires stoichiometric amounts of the protein. Addition of the human single-strand binding (SSB) holoprotein complex hRP-A reduces the requirement of HPP-1 in the reaction up to 20-fold. Although the combination of homologous pairing and SSB activities is similar to the bacterial strand-exchange process, the numbers, size, and requirements of the human reaction appear to preclude any detailed comparisons. We have used Z-DNA affinity chromatography as a major step in isolation of human recombination proteins and found that the activities appear to elute as a complex form in approximate multiples of 500 kDa. Associated with the homologous recombination complex is a potent blunt-end ligation activity that appears to mimic the nonhomologous joining functions that are frequently seen following transfection of DNA into mammalian cells. A simple scheme for the association of homologous and nonhomologous recombination functions in mammalian cells is discussed.
Fishel
R
R
Laboratory of Chromosome Biology, NCI-Frederick Cancer Research and Development Center, Maryland 21702.
Derbyshire
M K
MK
Moore
S P
SP
Young
C S
CS
D001483:Base Sequence; D002460:Cell Line; D002845:Chromatography; D004247:DNA / Q000378:metabolism; D011088:DNA Ligases / Q000378:metabolism; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D051716:Replication Protein A; D012441:Saccharomyces cerevisiae / Q000032:analysis / Q000235:genetics; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A; D004247:DNA; D015719:Single-Strand Specific DNA and RNA Endonucleases; D004264:DNA Topoisomerases, Type I; D011088:DNA Ligases