Overexpression of RAD51 suppresses recombination defects: a possible mechanism to reverse genomic instability

Abstract

RAD51, a key protein in the homologous recombinational DNA repair (HRR) pathway, is the major strand-transferase required for mitotic recombination. An important early step in HRR is the formation of single-stranded DNA (ss-DNA) coated by RPA (a ss-DNA-binding protein). Displacement of RPA by RAD51 is highly regulated and facilitated by a number of different proteins known as the 'recombination mediators'. To assist these recombination mediators, a second group of proteins also is required and we are defining these proteins here as 'recombination co-mediators'. Defects in either recombination mediators or co-mediators, including BRCA1 and BRCA2, lead to impaired HRR that can genetically be complemented for (i.e. suppressed) by overexpression of RAD51. Defects in HRR have long been known to contribute to genomic instability leading to tumor development. Since genomic instability also slows cell growth, precancerous cells presumably require genomic re-stabilization to gain a growth advantage. RAD51 is overexpressed in many tumors, and therefore, we hypothesize that the complementing ability of elevated levels of RAD51 in tumors with initial HRR defects limits genomic instability during carcinogenic progression. Of particular interest, this model may also help explain the high frequency of TP53 mutations in human cancers, since wild-type p53 represses RAD51 expression.

Figure 1.

Simplified schematic to depict the mediator step of recombination: displacement of RPA by RAD51. A critical step in homologous re-combinational repair is the displacement of RPA, the trimeric ssDNA-binding protein, by the RAD51 strand transfer protein. This step is highly regulated by cells to ensure that potentially deleterious events are avoided. Many different proteins are involved in assisting RAD51 to displace RPA at this stage, and the proteins that are directly involved are known as the ‘recombination mediators’ (Table 1). There are also additional proteins that function to assist the mediators or assist in their localization to DNA damage, and in this review, the proteins assisting the mediators are defined as ‘recombination co-mediators’. Human BRCA2 is a mediator that interacts directly with approximately eight RAD51 molecules and transports them to the site of ss-DNA bound by RPA, while PALB2 and BRCA1 are co-mediators that directly and indirectly, respectively, interact with BRCA2 and assist in localizing it, with RAD51 bound, to the sites of DNA damage (for references, see Table 1). DSB, double-strand DNA break; M/R/N, MRE11-RAD50-NBS1 complex.

Figure 2.

Effects of RAD51 overexpression in isogenic wild-type and BRCA1-deleted DT40 cells. (A) In cells with wild-type HRR, overexpression of RAD51 results in a reduced ability to repair cisplatin-induced DNA damage. Such increased sensitivity to a DNA cross-linking agent frequently coincides with genomic instability in untreated HRR-defective cells, presumably due to unrepaired spontaneous damage (16,17,44). (B) Alternatively, in cells with a BRCA1 homozygous deletion, the HRR defect is complemented by RAD51 overexpression. The complemented cells also presumably have increased genomic stability, a concept incorporated into Model I. Importantly, comparing just the cells overexpressing RAD51 (in both panels), the BRCA1 deletions actually result in increased resistance to cisplatin, and presumably also increased genomic stability, a concept incorporated into Model II. This figure is adapted directly from the data in Figure 2D of (24), with permission from Douglas K. Bishop and AACR.

Figure 3.

Schemes to explain the order of events in the two proposed models. (A) Model I: normal cells that develop an HRR defect (e.g. BRCA1^−/−) select for loss of TP53, which then acts to up-regulate RAD51 expression (darker nuclei represent higher levels of RAD51). Overexpression of RAD51 can in some cases lead to partial suppression of the original HRR defect, helping to stabilize the genome. (B) Model II: in some cancers, TP53 mutations are early events and these mutations may up-regulate RAD51 expression, resulting in increased genomic instability. Subsequent mutations/silencing in an HRR function partially suppresses the phenotype of RAD51 overexpression, reducing genomic instability. The asterisk indicates that for sporadic cancers many of these HRR mutations are likely to be in genes that exhibit haploinsufficiency, or the mutations themselves may be dominant-negative alleles.

Figure 4.

(A) Summary of Model I explaining cancer development initiated by HRR defects. The model suggests that many cancers with genomic instability are initiated by HRR defects, just as cancers with MSI are initiated by MMR defects. In cancers with HRR defects, selection for up-regulation of RAD51 acts to suppress the original HRR defect, partially restoring genomic stability and enhancing cell proliferation. TP53 mutations frequently result in the up-regulation of RAD51, and are selected for in cancers with a pre-existing HRR defect. HNPCC, hereditary non-polyposis colorectal cancer. (B) Summary of Model II explaining cancer development initiated by RAD51 up-regulation. In this model, inactivation of TP53 and up-regulation of RAD51 occur early in sporadic oncogenesis (i.e. cancers initiated by spontaneous non-inherited mutations) (left side). In some BRCA heterozygous cells (BRCA1^+/− or BRCA2^+/−), these events occur prior to functional loss of HRR resulting from a mutation or silencing of the second BRCA allele (right side). These are identical models, with the exception of the underlined phrases.