Physical Interaction between Human RAD52 and RPA Is Required for Homologous Recombination in Mammalian Cells*

The yeast RAD52 protein is essential for DNA double-strand break repair, and meiotic and mitotic recombination. RPA is a protein complex of three subunits (70, 34, and 11 kDa) that has been shown to be involved in DNA replication, nucleotide excision repair, and homologous recombination. Here, we demonstrate a physical interaction between human RAD52 and RPA in vivo and in vitro. In addition, the domain (amino acids 221-280) in RAD52 protein that mediates the interaction with the 34-kDa subunit of RPA was also determined. Overexpression of mutant RAD52 proteins lacking the interaction domain (amino acids 221-240, 241-260, and 261-280) failed to induce homologous recombination in monkey cells. We have previously shown that overexpression of human RAD52 induced homologous recombination in these cells. These results suggest that direct physical interactions between RAD52 and RPA are essential for homologous recombination in mammalian cells.

[1]  J. Hurwitz,et al.  Studies on the initiation and elongation reactions in the simian virus 40 DNA replication system. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[2]  R. Rothstein,et al.  A mutation in the gene encoding the Saccharomyces cerevisiae single-stranded DNA-binding protein Rfa1 stimulates a RAD52-independent pathway for direct-repeat recombination , 1995, Molecular and cellular biology.

[3]  F. Dean,et al.  Reconstitution of functional human single-stranded DNA-binding protein from individual subunits expressed by recombinant baculoviruses. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[4]  R. Legerski,et al.  An interaction between the DNA repair factor XPA and replication protein A appears essential for nucleotide excision repair , 1995, Molecular and cellular biology.

[5]  F. Dean,et al.  Simian virus 40 (SV40) DNA replication: SV40 large T antigen unwinds DNA containing the SV40 origin of replication. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[6]  Dong-Kyoo Kim,et al.  Role of the 70-kDa Subunit of Human Replication Protein A (I) , 1996, The Journal of Biological Chemistry.

[7]  P. Modrich,et al.  Mismatch repair, genetic stability, and cancer. , 1994, Science.

[8]  P. Berg,et al.  Complex formation in yeast double-strand break repair: participation of Rad51, Rad52, Rad55, and Rad57 proteins. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[9]  R. Wood,et al.  Mammalian DNA nucleotide excision repair reconstituted with purified protein components , 1995, Cell.

[10]  C. Ingles,et al.  RPA involvement in the damage-recognition and incision steps of nucleotide excision repair , 1995, Nature.

[11]  F. Dean,et al.  Replication of simian virus 40 origin-containing DNA in vitro with purified proteins. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[12]  P. Berg,et al.  A novel allele of Saccharomyces cerevisiae RFA1 that is deficient in recombination and repair and suppressible by RAD52 , 1995, Molecular and cellular biology.

[13]  R. Fishel,et al.  The human homologous pairing protein HPP-1 is specifically stimulated by the cognate single-stranded binding protein hRP-A. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[14]  J. Li,et al.  Initiation of simian virus 40 DNA replication in vitro: large-tumor-antigen- and origin-dependent unwinding of the template. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[15]  J. Stringer,et al.  Spontaneous and induced homologous recombination betweenlacZ chromosomal direct repeats in CV-1 cells , 1994, Somatic cell and molecular genetics.

[16]  D. Lane,et al.  Requirement for the replication protein SSB in human DMA excision repair , 1991, Nature.

[17]  B. Stillman,et al.  Yeast replication factor-A functions in the unwinding of the SV40 origin of DNA replication , 1989, Nature.

[18]  J. Hurwitz,et al.  Multiple functions of human single-stranded-DNA binding protein in simian virus 40 DNA replication: single-strand stabilization and stimulation of DNA polymerases alpha and delta. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[19]  Min-Seon Park.  Expression of Human RAD52 Confers Resistance to Ionizing Radiation in Mammalian Cells (*) , 1995, The Journal of Biological Chemistry.

[20]  M. Resnick.  6 – Investigating the Genetic Control of Biochemical Events in Meiotic Recombination , 1987 .

[21]  Dong-Kyoo Kim,et al.  Human Xeroderma Pigmentosum Group A Protein Interacts with Human Replication Protein A and Inhibits DNA Replication (*) , 1995, The Journal of Biological Chemistry.

[22]  E. Egelman,et al.  RecA-like recombination proteins in eukaryotes: functions and structures of RAD51 genes. , 1993, Cold Spring Harbor symposia on quantitative biology.

[23]  F. Dean,et al.  Binding and unwinding—How T antigen engages the SV40 origin of DNA replication , 1990, Cell.

[24]  Min-Seon Park,et al.  Specific Interactions between the Human RAD51 and RAD52 Proteins (*) , 1996, The Journal of Biological Chemistry.

[25]  B. Stillman,et al.  Cellular factors required for multiple stages of SV40 DNA replication in vitro. , 1988, The EMBO journal.

[26]  A. Yasui,et al.  DNA Repair Protein XPA Binds Replication Protein A (RPA) (*) , 1995, The Journal of Biological Chemistry.

[27]  A. Sancar.  Mechanisms of DNA excision repair. , 1994, Science.

[28]  A. Shinohara,et al.  Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein , 1992, Cell.

[29]  W. Heyer,et al.  An essential Saccharomyces cerevisiae single‐stranded DNA binding protein is homologous to the large subunit of human RP‐A. , 1990, The EMBO journal.

[30]  Suk-hee Lee,et al.  The Role of the 34-kDa Subunit of Human Replication Protein A in Simian Virus 40 DNA Replication in Vitro(*) , 1995, The Journal of Biological Chemistry.

[31]  L. Symington,et al.  Copyright � 1995, American Society for Microbiology Functional Differences and Interactions among the Putative , 1995 .

[32]  R. Jessberger,et al.  A mammalian protein complex that repairs double-strand breaks and deletions by recombination. , 1993, The Journal of biological chemistry.

[33]  F. Studier,et al.  Use of T7 RNA polymerase to direct expression of cloned genes. , 1990, Methods in enzymology.

[34]  T. Kelly,et al.  Purification and characterization of replication protein A, a cellular protein required for in vitro replication of simian virus 40 DNA. , 1988, Proceedings of the National Academy of Sciences of the United States of America.