The abundance of Rad51 protein in mouse embryonic stem cells is regulated at multiple levels.
DNA double-strand breaks (DSBs) in embryonic stem (ES) cells are repaired primarily by homologous recombination (HR). The mechanism by which HR is regulated in these cells, however, remains enigmatic. To gain insight into such regulatory mechanisms, we have asked how protein levels of Rad51, a key component of HR, are controlled in mouse ES cells and mouse embryo fibroblasts (MEFs). The Rad51 protein level is about 15-fold higher in ES cells than in MEFs. The level of Rad51 mRNA, however, is only ~2-fold higher, indicating that the differences in mRNA levels due to rates of transcription or mRNA stability are not sufficient to account for the large difference in the abundance of Rad51 protein. Comparison of Rad51 half-lives between ES cells and MEFs also did not explain the elevated level of Rad51 protein in the ES cells. A comparative assessment of the Rad51 translation level demonstrated that it is translated with much greater efficacy in ES cells than in MEFs. To determine whether this high level of translation in ES cells is a general phenomenon in these cells or whether it is a characteristic of specific proteins, such as those involved with recombination and cell cycle progression, we compared mechanisms that regulate the level of Pcna in ES cells with those that regulate Rad51. The half-life of Pcna and its rate of synthesis were considerably different from those of Rad51 in ES cells, demonstrating that regulation of Rad51 abundance cannot be generalized to other ES cell proteins and not to proteins involved in DNA replication and cell cycle control. Finally, we show that only a small proportion of the abundant Rad51 protein population is activated under basal conditions in ES cells and recruited to DNA DSBs and/or stalled replication forks.
Department of Molecular Genetics, University of Cincinnati, College of Medicine, Cincinnati OH 45267, USA. tichyed@ucmail.uc.edu
Elisia D
ED
Tichy
Resmi
R
Pillai
Li
L
Deng
Jay A
JA
Tischfield
Philip
P
Hexley
George F
GF
Babcock
Peter J
PJ
Stambrook
D000818:Animals; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair / Q000235:genetics; D004261:DNA Replication / Q000235:genetics; D050684:E2F Transcription Factors / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology; D053595:Embryonic Stem Cells / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D005786:Gene Expression Regulation; D051379:Mice; D018809:Proliferating Cell Nuclear Antigen / Q000096:biosynthesis; D014176:Protein Biosynthesis; D055550:Protein Stability; D051135:Rad51 Recombinase / Q000235:genetics / Q000378:metabolism*
D050684:E2F Transcription Factors; D018809:Proliferating Cell Nuclear Antigen; D051135:Rad51 Recombinase; C495154:Rad51 protein, mouse