Expression and regulation of Rad51 in human cells

Abstract

Double strand breaks (DSBs) can be caused by exogenous DNA damaging agents or by endogenous processes, and may lead to chromosomal breakage and rearrangement resulting in apoptosis or tumorigenesis. The repair of DSBs in higher eukaryotes is to a large extent accomplished by homologous recombination (HR), a process in which DNA from a complementary strand is used as a template for repair synthesis. This thesis focuses on the expression and regulation of human Rad51 (HsRad51), the key component of a protein complex involved in HR.

1) The expression of the HsRad51 gene during the cell cycle was studied in mitogen stimulated primary human peripheral blood lymphocytes (PBLs). A peak in the HsRad51 mRNA and protein expression was observed in S/G2. Studies with inhibitors of DNA, RNA and protein synthesis indicated transcriptional regulation of the gene and coregulation of the expression with the S-phase.

Treatment of PBLs with high doses of genotoxic agents resulted in decreased HsRad51 expression. The protein was shown to be cleaved, producing a 33 kD fragment, during drug- and receptor mediated apoptosis. The time-course of the cleavage coincided with inter-nucleosomal DNA fragmentation and with cleavage of human poly (ADP-ribose) polymerase. The absence of cleavage in MCF-7 cells showed that caspase-3 is essential for HsRad51 cleavage in vivo. Recombinant caspase-3 cleaved in vitro translated HsRad51 and endogenous HsRad51 in Jurkat cell extracts. The cleavage site was mapped to AQVD274¯G. This site is located in a region of the HsRad51 protein sequence that has been shown to be important for the interaction with Rad52. Abrogation of the interaction may lead to decreased activity of HsRad51.

Inducible, stable transfectants were generated in order to study the effects of acute HsRad51 overexpression. Induction of HsRad51 protein expression resulted in a dose-dependent decrease in plating efficiency and growth rate. An accumulation of HsRad.51 overexpressing cells in the G2 phase of the cell cycle was observed following release from synchronization. The fraction of apoptotic cells increased with the time of overexpression. Using an array hybridization technique, increased mRNA levels of a set of genes involved in cell cycle regulation, survival and apoptosis was observed in the HsRad51 overexpressors. A selection of cells resistant to the observed negative effects of acute HsRad51 overexpression on end-points related to cell proliferation could conceivably be of importance for tumour progression.

In summary, the results show that HsRad51 expression is coregulated with the cell cycle, confirming a role of the protein in HR between sister chromatids. The cleavage of HsRad51 by caspase-3 indicates that HsRad51 belongs to a group of repair proteins which are specifically cleaved during the execution phase of apoptosis. The observed detrimental effects of acute HsRad51 overexpression may be of importance in carcinogenesis.